RU2253116C2 - Retentive chromatography method for separating analytes in sample - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves producing substrate for detecting analytes in a sample by treating the sample at least under two selectivity conditions depending on adsorbent and eluate combinations. It enables one to retain analyte with the sorbent, to make identification by applying desorbing spectrometry. Method for setting diagnosis with the substrate is given.

EFFECT: high specificity of diagnosis method.

34 cl, 33 dwg

Description

FIELD OF THE INVENTION

The present invention relates to the field of separation technologies and analytical biochemistry.

The methods of the present invention can be used in biology and medicine, including gene function analysis, differential gene expression, protein detection, cell and clinical diagnosis and screening of new drugs.

Cell function, both normal and pathological, is at least partially dependent on the genes expressed in the cell (i.e., gene function). Gene expression has both qualitative and quantitative aspects. That is, cells can differ both in the set of specific expressed genes and in the relative level of expression of the same gene. Differential gene expression may occur, for example, in different expression of the proteins encoded by the gene, or in post-translational modifications of the expressed proteins. For example, proteins can be modified with carbohydrate or phosphate groups or undergo cutting. Thus, at the biochemical level, a cell is a complex mixture of organic biomolecules.

One of the goals of functional genomics (“proteomics”) is the identification and characterization of organic biomolecules, which are differentially expressed in various types of cells. By comparing expression, molecules can be identified that may be responsible for a particular pathological activity of the cell. For example, the identification of a protein expressed in cancer cells, but not in normal cells, can be useful in the diagnosis and, ultimately, for the selection of drugs and treatment of pathology. Upon completion of the Human Genome program, all human genes will be cloned, sequenced, and information about them will be compiled into databases. In this "post-genomic" world, the ability to identify differentially expressed proteins will, in turn, lead to the identification of the genes that encode them. Thus, the power of genetics will lead to the solution of cell function problems.

Differential chemical analyzes of gene expression and their functions require tools that can separate a complex mixture of molecules in a cell, identify and quantify them, even when they are present in trace amounts. However, the methods of analytical chemistry existing for these purposes are limited for each of the listed problems. One of the popular methods for separating biomolecules is electrophoresis. Often the first separation of proteins by isoelectric focusing in a gel is combined with the second separation by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS-PAGE). The result is a map on which proteins are separated by isoelectric point (total charge) and size (i.e. mass). However, being useful, this method has several limitations. Firstly, using this method, information can only be obtained on two characteristics of a biomolecule — mass and isoelectric point (“pI”). Secondly, the resolution at each separation is limited by the resolution of the gel. For example, it is often difficult to separate molecules whose mass differs by less than 5%, or the isoelectric point differs by less than 0.5 pI. Thirdly, the load on the gel is limited and, accordingly, sensitivity is limited; often it is not possible to detect biomolecules expressed in small quantities. Fourth, small proteins and peptides with a molecular mass of less than 10-20 kD are insoluble by this method.

Other analytical methods can overcome one or more of these limitations, but it is rather difficult to combine them effectively. For example, analytical chromatography can separate biomolecules based on a variety of analyte / adsorbent interactions, but multivariate analysis is difficult and time consuming. In addition, these methods have limited sensitivity.

Clinical diagnosis requires specific detection of known disease markers. However, the development of such diagnostic approaches is hampered by the need to obtain reagents that specifically bind to markers or are able to discriminate a marker in a complex mixture.

When developing new drugs, agents that modulate ligand / receptor interactions must be rapidly screened. Often the limiting step in such screening is the step of detecting ligand / receptor interactions. Therefore, quick and specific methods for identifying the binding event will have significant advantages.

Until recently, the process of identifying a potential marker or member of a ligand / receptor pair to produce an agent that specifically binds to a marker or ligand / receptor has been very difficult. According to one approach, normal and damaged (diseased) tissues are compared in order to identify mRNA species or expressed marker sequences (“EST”) whose level is increased or decreased in the damaged tissue. These types of RNAs are isolated and the polypeptides encoded by them are obtained using routine recombinant DNA methods. The polypeptides are then isolated and used as immunogens to produce marker-specific antibodies. Antibodies can be used, for example, in an ELISA to determine the amount of marker in a sample taken from a patient.

This process is long and tedious. To obtain such antibodies, it takes from nine months to a year, while no less time is required to develop protocols for the isolation of such an amount of polypeptide that is sufficient for immunization. In addition, the method is based on the conclusion that differences in RNA expression are reflected as differences in protein expression. However, this conclusion is not always true. Therefore, the methods by which directly differentially expressed proteins can be directly detected and by which specific ligands can be obtained in a much shorter time will have significant advantages.

Thus, analytical biochemistry, biology, and medicine require methods and tools for separating complex mixtures of organic biomolecules, identifying individual biomolecules in a mixture, and identifying specific molecular recognition events involving one or more target analytes.

SUMMARY OF THE INVENTION

The present invention describes devices and methods for retentate chromatography. Retentate chromatography is a combinatorial method of highly informative separation of analytes in complex mixtures through the use of multidimensional separation methods. It provides reliable analyte detection and functional analysis of applicability for biology and medicine, which is achieved on the basis of a single integrated operating system for direct detection of the expression pattern of analyte associated with gene function, protein function, cell function and body function as a whole.

More specifically, analytes can be resolved in various two-dimensional formats, and multidimensional information becomes available. First, analytes are separated based on their ability to adsorb to the stationary phase under at least two different conditions of selectivity, such as anion / cationic potential, hydrophobicity / hydrophilicity or specific biomolecular recognition. The analytes are then separated in another dimension based on their mass using desorption spectrometry (i.e., laser desorption mass spectrometry), which makes it possible to detect separated analytes. The nature of the adsorbent onto which analytes are adsorbed provides information on the physicochemical properties of the analyte.

Thus, the invention claims a tool for molecular detection and diagnostics, combining the capabilities of both parallel and multiplex processing of analytes. Since analytes are detected directly, the invention enables the simultaneous transfer of signals from two or more independent target analytes from one "cell" (ie, a predetermined chip localization) during one operational cycle.

Retentate chromatography differs from conventional chromatography in several ways. First, retent chromatography reveals analytes that are retained on an adsorbent. In conventional chromatography methods, analytes elute from the adsorbent before detection. For conventional chromatography, the detection of an analyte that is not eluted from the adsorbent is not a routine or routine approach. Thus, retentate chromatography provides direct information on the chemical or structural characteristics of the retained analytes. Secondly, the combination of adsorption chromatography with detection by desorption spectrometry provides extremely high sensitivity in the femtomolar range and an unusually fine resolution. Thirdly, partly due to the direct detection of analytes, retentate chromatography makes it possible to quickly analyze retentates (retained analytes) in many different conditions of selectivity, thereby ensuring a fast multidimensional characterization of analytes in the sample. Fourth, adsorbents can be attached to the substrate in a series of predefined locations. This allows parallel processing of analytes deposited on different spots of the adsorbent (ie, "affinity sites" or "spots") on the matrix under various elution conditions.

Retentate chromatography has many uses in biology and medicine. These include combinatorial biochemical separation and purification of analytes, the study of differential gene expression and molecular recognition events, diagnosis and screening of potential drugs.

One of the main applications of retentate chromatography as an analytical tool involves bringing a sample into contact with a combinatorial set of various adsorbent / eluant combinations and determining the behavior of the analyte under various conditions. This achieves the purification of the analyte and the determination of the conditions necessary for the detection of the analyte in the sample. Substrates with adsorbents identified in this way can serve as specific analyte or analyte detectors. In the sequential extraction method, the sample is brought into contact with the first adsorbent / eluant combination and washed, the analytes adsorbed by the first adsorbent are depleted and contacted with the second adsorbent to deplete other analytes. The selectivity conditions under which the analytes are retained can also be used in preparative purification procedures when the crude sample containing the analyte is subsequently contacted with the adsorbents that hold it, the impurities are removed, and the retained analyte is collected from the adsorbent for subsequent cycles.

One aspect of the invention is that each class or type of molecular recognition event (ie, adsorbent-target: analyte-target interaction) is characterized by a specific selectivity condition within a predetermined localization on the matrix and is detected directly while the bound molecules are still localized (ie, "held") in a predetermined localization. Thus, selection and detection do not require elution, isolation, amplification or labeling of the target analyte.

Another aspect of the present invention is that the detection of one or more molecular recognition events in one or more predetermined locations on the matrix does not require the removal or use of more than a small fraction of the total adsorbent analyte. Thus, an unused portion can be investigated further during one or more cycles of “secondary processing” conducted directly in situ (ie, within a predetermined location) for the purpose of clarifying the structure and function, including subsequent assembly, disintegration, modification or amplification (direct or indirect).

In an interactive process, referred to as "sequential resolution", adsorbents with enhanced analyte specificity can be obtained. Moreover, adsorbents or eluants, for which the analyte retention capacity has already been shown, are tested with additional variables to determine combinations with better binding characteristics. Another method makes it possible to quickly create substrates with antibody adsorbents specific for the analyte. The method involves binding the analyte to an adsorbent and screening a phage library to detect phage that binds the analyte.

Retentate chromatography also has applications in molecular and cellular biology. Analytes that are differentially present in two samples (e.g., differentially expressed proteins in two cell extracts) can be identified by contacting the samples with various adsorbent / eluant combinations followed by analysis by desorption spectrometry. At the same time, a highly informative resolution of the system is realized, which is unattainable in other separation and detection systems. Unknown target proteins can be identified by determining the physicochemical characteristics, including molecular weight, based on the chemical characteristics of the adsorbent / eluant combination. The information obtained can be used in screening databases to search for proteins with similar profiles.

Separation biochemistry methods and adsorbents developed in accordance with the claimed methods are applicable in the diagnosis. More specifically, both chemical and biospecific adsorbents can be developed to detect important diagnostic markers. In certain embodiments, the substrate may comprise an adsorbent stain matrix, suitably selected combinations of markers, diagnostic for a given disease or syndrome.

Retentate chromatography is useful in screening for potential drugs. One of the members of the receptor / ligand pair is attached to the adsorbent and its ability to bind a partner is tested in the presence of an agent. Due to the speed with which adsorption can be established, it is easy to test combinatorial libraries of agents for their ability to modulate interaction.

As one aspect of the invention, a method for highly informative separation of at least one analyte in a sample is claimed. This method is a combinatorial method, including parallel separation and detection of multiple analytes. The method includes the following steps: a) processing the analyte in at least two conditions of selectivity, with each condition of selectivity being determined by a combination of adsorbent and eluant, which makes it possible to retain the analyte with an adsorbent; and b) detecting an analyte held under various conditions of selectivity using desorption spectrometry. Detection of an analyte held under various conditions of selectivity provides highly informative analyte separation.

According to one embodiment of the invention, each different selectivity condition is determined at a different predetermined location for parallel processing. Another option provides a method comprising the following steps: i) processing the analyte under the first condition of selectivity in a certain location, which creates conditions for the retention of the analyte with an adsorbent; ii) detecting analyte retained under the first condition of selectivity by desorption spectrometry; iii) washing the adsorbent under a second, different condition of selectivity at a certain location, which creates conditions for the retention of the analyte by the adsorbent; iv) detection of retained analyte by desorption spectrometry.

In one embodiment, the analyte is an organic biomolecule, a multimeric molecular complex, or a macromolecular complex. In another embodiment, the organic biomolecule is an enzyme, an immunoglobulin, a cell surface receptor, or an intracellular receptor.

According to one embodiment of the invention, the adsorbent is an anion, a cation, an adsorbent of hydrophobic interaction, a polypeptide, a nucleic acid, a carbohydrate, lectin, a dye, a reducing agent, a hydrocarbon, or a combination thereof. According to another embodiment of the invention, the adsorbent is attached to a substrate comprising glass, ceramic, magnetic material, an organic polymer, a conductive polymer, a natural biopolymer, a metal or a metal coated with an organic polymer. According to another embodiment of the invention, the adsorbent is in the form of a microemulsion, latex, layer or bead. According to another embodiment of the invention, the substrate is arranged in a line or a rectangular matrix. According to another embodiment of the invention, the adsorbents are located on the substrate at various locations before the analytes are subjected to selectivity conditions. According to another embodiment of the invention, the adsorbents are located on the substrate at various locations after the analytes are subjected to selectivity conditions. According to another embodiment of the invention, the various selectivity conditions are different binding conditions or different elution conditions.

According to one embodiment of the invention, the detection step comprises determining the analyte mass using laser desorption mass spectrometry.

According to another embodiment of the invention, the selectivity conditions are selected so as to optimize the retention of the analyte by the adsorbent. According to another embodiment of the invention, the selectivity conditions are determined for various adsorbents and one eluant.

Another embodiment of the invention includes the step of producing a substrate containing adsorbents in predetermined locations, with each adsorbent meeting the selectivity condition defined for analyte retention. According to another embodiment of the invention, the elution conditions differ with respect to pH, buffer capacity, ionic strength, water structure characteristics, type of detergent, detergent strength, hydrophobicity or dielectric constant. According to another embodiment of the invention, many selectivity conditions are determined by the same eluant.

According to another embodiment of the invention, a method for sequential extraction of analytes from a sample is claimed. This is a combinatorial sequential method for separating and purifying many analytes in parallel. The method includes the steps of: a) processing the sample in the first condition of selectivity to retain analytes with the first adsorbent to obtain an unstoppable sample; b) collecting an unstoppable sample containing analytes, treating the unstoppable sample in a second selectivity condition to retain the analytes with a second adsorbent to obtain an unsupervised sample; c) detection of marker analyte selectivity retained under various conditions by desorption spectrometry.

As another aspect of the invention, a substrate for desorption spectrometry is disclosed, comprising an adsorbent with binding characteristics that vary in a gradient along one or more linear axes.

Another aspect of the present invention is a method for sequentially identifying a selectivity condition with improved analyte resolution in a sample. The method includes the steps of: a) identifying a selectivity condition under which the analyte contained in the sample is retained by i) processing the sample in a set of selectivity conditions, each selectivity condition being determined by at least one binding characteristic and at least one elution characteristic; ii) detection by desorption spectrometry of the analyte retained in each selectivity condition; (iii) identification of the selectivity condition in which the analyte is held; and b) identifying the selectivity condition with improved analyte resolution by i) selecting at least one binding characteristic or elution characteristic from the identified selectivity condition and introducing it into a permanent set of selectivity characteristics; ii) processing the sample in a modified set of selectivity conditions, while each selectivity condition in the modified set includes (1) selectivity characteristics from a constant set and (2) a binding or elution characteristic not included in the constant set; and (iii) identification by desorption spectrometry of the selectivity condition from the modified kit, which provides retention of the analyte with a higher resolution than the previously identified selectivity condition. According to one embodiment of the invention, at this stage, step b) is repeated at least once. According to another embodiment of the invention, step b) is repeated until a selectivity condition is identified in which only the target analyte is retained from the sample.

Another aspect of the present invention is a substrate for desorption spectrometry containing an adsorbent from selectivity conditions that resolve the analyte by sequential resolution. According to one embodiment of the invention, the substrate is supplied in the form of a kit containing an eluant from the condition of selectivity, or instructions for using the eluant in combination with an adsorbent.

Another aspect of the present invention is a method for preparative purification of an analyte from impurities contained in a sample. The method includes the following steps: a) bringing the sample into contact with the substrate under a variety of different selectivity conditions; detection of analyte selectivity retained in a variety of different conditions by desorption spectrometry; identification of the selectivity conditions under which the analyte is held; b) purification of the analyte by repeating for a variety of different identified conditions of selectivity selectivity sequential steps, including i) bringing the sample into contact with the adsorbent in the identified condition of selectivity, which ensures retention of the analyte by the adsorbent; ii) separating the analyte from impurities that are not retained by the substrate; and iii) collecting analyte from the adsorbent.

Another aspect of the present invention is a method for producing a substrate for detecting at least one analyte in a sample. This method is a combinatorial method for the construction and identification of analyte-specific adsorbents. It is useful for detecting target analytes. The method includes the following steps: a) processing the sample under at least two different conditions of selectivity, each condition of selectivity being determined by a combination of adsorbent and eluant and makes it possible to retain the analyte with an adsorbent; b) identification by desorption spectrometry of at least one selectivity condition under which the analyte is curved; and c) preparing a substrate containing at least one adsorbent from the identified selectivity condition. According to one embodiment of the invention, the identification step comprises identifying at least one selectivity condition under which a plurality of analytes are curtailed. According to another embodiment of the invention, the step of obtaining a substrate involves the production of a plurality of adsorbents that retain the analyte under a certain selectivity condition, acting as a multiplex adsorbent.

Another aspect of the present invention is a diagnostic method in a subject of a disease for which at least one marker is characteristic. This is a combinatorial method for the simultaneous detection of multiple diagnostic markers. This method includes the following steps: a) obtaining a substrate for desorption spectrometry, which contains at least one predetermined localization, with each predetermined localization containing an adsorbent that resolves at least one diagnostic marker under a certain elution condition; b)

bringing the substrate into contact with a biological sample taken from a subject in an elution condition that ensures retention of the diagnostic marker; and c) detecting a retained diagnostic marker by desorption spectrometry. Detection of a retained diagnostic marker provides a diagnosis of the disease.

Another aspect of the present invention is a kit for detecting analyte in a sample, comprising (1) a substrate useful in desorption spectrometry, containing at least one predetermined localization, each predetermined localization contains an adsorbent that resolves the analyte under the condition of selectivity determined by the adsorbent and eluant, and (2) an eluant or sample processing instructions subject to selectivity. According to one embodiment of the invention, the kit is characterized by a plurality of diagnostic markers and the substrate contains a plurality of predetermined locations, with each predetermined location containing an adsorbent that resolves at least one diagnostic marker.

Another aspect of the present invention is a substrate useful in desorption spectrometry, containing at least one adsorbent in at least one predetermined location, while at least one adsorbent resolves many diagnostic markers of the pathological condition in a sample taken from a patient.

Another aspect of the present invention is a method for selecting candidates for identity to an analyte protein. This is a combinatorial method for identifying proteins based on at least two physicochemical properties. The method includes the following steps: a) determining a set of coinciding parameters that determine at least the first and second physicochemical properties of the analyte protein in the sample by i) bringing the analyte into contact with many different selectivity conditions in which the adsorption of the analyte protein by the substrate occurs on the basis of affinity due to the physicochemical property of the analyte protein; and ii) detecting analyte selectivity retained under various conditions by desorption spectrometry; and b) the implementation on a programmable digital computer of the following

stages: i) access to a database containing for each member of the set of reference polypeptides a set of parameters that define at least the first and second physicochemical properties of reference polypeptides; ii) the introduction of a set of parameters that determine the physicochemical properties of the analyte protein; iii) sorting from a database of reference polypeptides having the same set of parameters. Sorted reference polypeptides are candidates for protein analyte identity. Unsorted reference polypeptides are excluded from candidates for identity.

Another aspect of the present invention is a method for sequential retention of analytes. This method is a method for monitoring multimeric macromolecular or supramolecular assembly. It is applicable for screening potential drugs for molecular recognition interference. The method includes the following steps: a) bringing the first sample into contact with the primary adsorbent and eluant, which makes it possible to retain the first analyte with adsorbent, and detect the adsorbed analyte by desorption spectrometry, while the retained first analyte becomes a secondary adsorbent; b) bringing the second sample into contact with the secondary adsorbent and detecting the adsorbed second analyte by desorption spectrometry, while the retained second analyte becomes a quaternary adsorbent.

Another aspect of the present invention is the detection of an enzyme in a sample. This method includes the following steps: a) obtaining a solid phase containing an adsorbent and an enzyme substrate attached to the adsorbent, wherein the activity of the enzyme with respect to the enzyme substrate leads to the formation of a product with a characteristic molecular weight; b) bringing the substrate into contact with the sample; and c) product detection using desorption spectrometry. Product detection means enzyme detection.

Another aspect of the present invention is a method for determining the differential presence of an analyte (i.e., differential expression) in a first and second biological sample. The method is applicable as a combinatorial method for monitoring differential gene expression by differential identification of proteins. The method includes the following steps: a) compilation of the first retention chart for the analyte in the first sample under at least one condition of selectivity; b) compiling a second retention chart for the analyte in the second sample under the same condition of selectivity; and c) detecting differences between the first and second retention cards. Differences in retention charts make it possible to establish the fact of the differential presence of the analyte in the first and second samples.

According to one embodiment of the invention, the method serves to establish the fact of differential expression of the protein in two different cells, and the first and second samples are cells or cellular material. In another embodiment, the purpose of the method is to establish that the agent has altered protein expression in a biological sample and the method includes the step of introducing the agent into the first biological sample, but not into the second biological sample. According to another embodiment, the first biological sample is obtained from a healthy subject, and the second biological sample is from a subject suffering from any disease. A sample may be, for example, blood, urine, serum or tissue. Those analytes that are found in increased amounts in samples obtained from patients are possible diagnostic markers. Typically, confirmation of a diagnostic marker involves its detection in many subjects.

Another aspect of the present invention is a method for identifying a ligand to a receptor. The method includes the following steps: a) obtaining a substrate containing an adsorbent, while the receptor is bound to the adsorbent; b) bringing the bound receptor into contact with a ligand-containing sample under conditions that make binding of the receptor and ligand possible; and c) detecting the bound ligand by desorption spectrometry.

Another aspect of the present invention is a screening method for determining that the agent modulates the binding between the target analyte and the adsorbent. This is a combinatorial screening method for potential drugs. The method includes the following steps: a) obtaining a substrate containing an adsorbent, despite the fact that the target analyte binds under elution conditions; b) bringing the substrate into contact with the target analyte and the adsorbent; c) determination of the level of binding between the target analyte and adsorbent desorption spectrometry; and d) establishing the fact that a certain level differs from a control level of binding when the substrate is brought into contact with a target analyte under elution conditions without an agent. The difference between the experimentally determined level and the control level indicates that the agent modulates binding.

Another aspect of the present invention is a method for detecting a genotype comprising a polynucleotide that encodes a polypeptide agent that specifically binds a target adsorbent. On the one hand, this is a combinatorial method for selecting an analyte-specific phage from the library, including the use of target proteins isolated by retentate mapping, or target proteins obtained in situ by transcription and in vitro translation. The method includes the following steps: a) obtaining a substrate containing a target adsorbent; b) obtaining a library containing many different genotypes, wherein each different genotype (organism) contains a polynucleotide in which a nucleotide sequence encoding a polypeptide agent is represented, and each different genotype (organism) has a surface on which the encoded polypeptide agent is represented; c) bringing the substrate into contact with the library under such elution conditions that make it possible to specifically bind the polypeptide agent to the target adsorbent, while the genotype (organism) containing the polypeptide agent is retained on the substrate; and d) detection of genetic material held on a substrate by desorption spectrometry.

According to one embodiment of the invention, the library is a phage library. According to another embodiment of the invention, the phage is phage M13. In yet another embodiment, the polypeptide is a single chain antibody. According to another embodiment of the invention, the target analyte is a polypeptide analyte that is differentially expressed in cells of various phenotypes. According to another embodiment of the invention, the substrate is a cell or a cell membrane.

According to one embodiment of the invention, the step of obtaining a substrate containing an adsorbent target includes the following steps: i) obtaining a substrate containing an adsorbent, wherein the adsorbent holds the analyte target in an elution condition; and ii) bringing the adsorbent into contact with the target analyte under elution conditions where retention of the target analyte by the adsorbent is possible, wherein the target analyte becomes the target adsorbent. According to another embodiment of the invention, the target analyte is a target polypeptide and step ii) comprises producing the target polypeptide in situ on the adsorbent by in vitro translation of the polynucleotide encoding the target polypeptide, and may also include amplification of the polynucleotide sequence in situ on the substrate.

According to another embodiment of the invention, the substrate comprises (1) an adsorbent that binds an anchor polypeptide and (2) at least one target organism, on the surface of which an anchor polypeptide and a target polypeptide acting as an adsorbent are presented. The target organism contains a polynucleotide that encodes a target adsorbent, wherein the target organism is coupled to the adsorbent via an anchor polypeptide.

According to another embodiment of the invention, the method further includes any of the following steps: sequencing the nucleotide sequence encoding the polypeptide agent; isolating a retained genotype or obtaining a polypeptide agent.

Another aspect of the present invention is a substrate for desorption spectrometry, consisting of an adsorbent that binds an anchored polypeptide present on the surface of the body, while the target polypeptide is also presented on the surface of the body, and the genotype of this organism contains a polynucleotide having a nucleotide sequence that encodes a polypeptide target.

Another aspect of the present invention is a method for detecting translation of a polynucleotide. The method includes the following steps: a) obtaining a substrate containing an adsorbent for desorption spectrometry; b) bringing the substrate into contact with the polynucleotide encoding the polypeptide and with agents for translating the polynucleotide in vitro, as a result of which the polypeptide is formed; c) treatment of the substrate with an eluant, which makes it possible to retain the polypeptide with an adsorbent; d) detection of the retained polypeptide by desorption spectrometry. Detection of a polypeptide provides confirmation of polynucleotide translation.

Brief Description of the Drawings

Figure 1 shows a substrate containing a lot of adsorbing spots in the form of a strip. The strip contains six different sets of adsorbents, classified on the basis of interaction forces (hydrophobic, ionic, coordinate covalent and mixed functions). The strip contains several spots of each type of adsorbent, which makes it possible to treat spots at different times with various eluants or archiving and subsequent analysis.

Figure 2 shows a rectangular matrix of adsorbents (potentials of surface interaction) in predetermined localizations. The matrix may also be in the form of a plate. The matrix contains various adsorbents. Upon contact with the analyte, each strip can be washed with various eluants (selectivity threshold modifiers). The retention analysis for various conditions of selectivity gives a retention map or recognition profile.

Figure 3 presents the quantitative analysis of analytes by desorption of the analyte in these localizations of the matrix and the quantitative determination of the desorbed analyte by laser desorption mass spectrometry.

On figa shows a computer system 1, which includes a monitor 3, a screen 5, a housing 7, a keyboard 9 and a mouse 11. The mouse 11 may have one or more buttons, such as buttons 13. In the housing 7 are CD-ROM 15 and hard a disk (not shown) that can be used to store and recall computer programs, including code, necessary to implement the present invention. While CD-ROM 17 is shown as the information medium, other media can also be used, including floppy disks, DRAM, hard disks, flash memory, tapes, etc. The case 7 also contains such usual computer components (not shown), like processor, memory, etc.

FIG. 4B shows a block diagram of a computer system 1 used to implement programs necessary for analyzing data obtained during the implementation of the present invention. As in FIG. 4A, computer system 1 includes a monitor 3 and a keyboard 9. Computer system 1 also includes subsystems such as a central processor 102, system memory 104, I / O controller 106, display adapter 108, removable disk 112, fixed disk 116, a network interface 118 and a speaker 120. The removable disk 112 may be any removable storage medium, such as floppy disks, cassettes, CD-ROMs, removable hard drives, flash memory, and the like. The fixed disk 116 may be an internal hard disk, DRAM, or the like. Other computer systems suitable for the purposes of the present invention may include additional subsystems or contain fewer such subsystems. For example, another computer system may comprise more than one processor 102 (i.e., a multiprocessor system) or cache memory.

5A-5F show retention charts for lysozyme under selective conditions, including six different adsorbents and several different eluants.

On figa-6B shows the resolution of low molecular weight and high molecular weight analytes in human serum on an adsorbent with immobilized metal.

On figa-7B shows the resolution of low molecular weight and high molecular weight analytes in human serum on different adsorbents with the same eluant.

On figa-8B shows the resolution of low molecular weight and high molecular weight analytes in the urine of a premature baby on various adsorbents using water as eluant.

Figure 9 shows the resolution of analytes in the urine of a premature baby using a hydrophobic phenyl adsorbent and three different eluants, while selective retention of one of the analytes (*) was detected when washing with Tween.

On figa-10D shows the resolution of the analytes in the culture medium of the cells of two different lines of breast cancer.

Figure 11 shows a composite retention map of the urine of a premature baby treated under selective conditions determined for six different adsorbents and three different eluants.

On Fig shows the separation in two-dimensional polyacrylamide gel (pI and apparent molecular weight) of the urine of a premature baby.

13 shows a method for panning phage libraries to isolate phage with a surface protein that specifically binds a target analyte. The substrate shown above shows that even several specifically binding phages can be detected by desorption spectrometry by detecting many surface proteins that contain the phage. Below is a substrate with several spots of adsorbent, designed in such a way that specifically targets the analyte. The phage is applied to the spots. Bound phage are detected by desorption spectrometry. Phage associated with another spot can be isolated and raised.

On Fig shows a ligand agent, in this case, a single chain antibody identified by the method of panning, which can be used as an adsorbent for retention of the target protein and subsequent study of protein-protein interactions. The target is purified in situ (spot 2) and used to pan the phage library (spot 4). A single chain antibody is isolated and attached to a substrate (spot 6) as an adsorbent. The target is then adsorbed by a single chain antibody. Now the target is held for the study of protein-protein interactions (spot 8).

FIG. 15 shows a method for screening potential drugs for the ability to interfere with protein binding to a ligand, in this case a single chain antibody. A single chain antibody specific for the target protein is held on the spot of the substrate by, for example, an antiphage antibody, which in turn can be held by protein A or protein G. The single chain antibody is contacted with the target protein and potential drug. The ability of the drug to bind a protein analyte and interfere with ligand binding by an analyte is controlled by desorption spectrometry.

Figure 16 shows a method for screening potential drugs for their ability to interfere with protein binding to a ligand. The method is similar to that shown in the previous drawing with the difference that desorption spectrometry controls the ability of the drug to interfere with analyte binding by ligand binding.

On Fig shows a method of screening potential drugs for the ability to interfere with the binding of the target protein (Protein target 1) with a secondary ligand (Protein target II). As in the previous two figures, the target held onto the substrate becomes the ligand adsorbent. In this case, the analyte is retained by a single chain antibody. The target is brought into contact with the target protein and potential drug. The ability of a drug to interfere with binding between an analyte and a ligand (e.g., binding an analyte target) is controlled by desorption spectrometry.

On Fig presents a diagram of the whole process, which begins with the identification of differentially expressed mRNAs or polypeptides and ends with the creation of a diagnostic platform for specific binding of polypeptides with subsequent detection by desorption spectrometry.

19A-19D show a retention map of a Hemophilus lysate on an adsorbent matrix. Figa: antionic adsorbent; figv: normal phase adsorbent; figs: Ni (II) adsorbent; fig.19D: hydrophobic adsorbent.

On figa-20C shows the sequential resolution of the analyte in the lysate of Hemophilus. The adsorbent in each case was an anionic adsorbent. Figa: in the first stage, after applying the sample, the spot was washed with 150 μl of 20 mm sodium phosphate, 0.5 M NaCl, pH 7.0. At the second stage, an adsorbent and a characteristic of sodium phosphate in the eluant were added to a constant set of characteristics. A new elution profile has been introduced. FIG. 20B: in the presence of 20 mM sodium phosphate, pH 7.0, the spot was washed with 0.05% Triton X100 and 0.15 M

NaCl (total 150 μl). Fig. 20C: in the presence of 20 mM sodium phosphate, pH 7.0, the spot was washed with 100 mM imidazole, 0.15 M NaCl (total 150 μl).

On figa-21D shows the results of a comparison of the components of the serum of normal and sick people. FIG. 21A: Retention map of normal serum at the Cu (II) site of the adsorbent matrix. Figv: Retention map of the serum of the patient at the site of Cu (II) adsorption matrix. 21C: Retained analytes from both serum samples are matched by superposition. To simplify the picture, each peak of the retained analyte is represented by a straight line, with dashed lines represent analytes retained from normal serum, and bold lines represent analytes retained from patient serum. Fig.21D: To more clearly identify the differences between the two samples, a comparison graph was constructed that shows the calculated ratios of retained analytes in the samples. For two analytes, marked with "*", a significant increase in the serum of patients was noted (an excess of 5-10 times).

On figa-22D shows a comparison of retention maps of the urine of the control, patient and treated with the drug mice on the Cu (II) adsorbent and quantitative determination of the marker in the urine of the patient and the treated mice.

On figa-23D shows retention maps of analytes in the urine of cancer patients, shown in the "gel" format. On the differential maps of patients 1, 2 and 3, two general analytes are visible, which are present in these patients in increased quantities.

On figa-24E shows the detection of phage M13 by desorption mass spectrometry by detecting the surface protein of gene VIII. Dilutions of the stock phage stock solution with a particle content of 10 ¹² per ml ranged from 1:10 to 1: 10,000,000,000.

On figa-25B shows the detection of retained phage M13 by desorption spectrometry using an anti-M13 antibody as adsorbent. On figa shows the retained phage M13, and the peaks represent the proteins of gene VIII and gene III. On figv shows the control peaks of the antibody adsorbent (single and double charged).

On figa-26D shows the adsorption of the adsorbent with the tat protein of the phage M13 carrying a single chain anti-tat antibody. Single peaks were obtained from phage dilutions from 1:10 to 1: 10,000.

On figa-27B shows the retention maps of TGF-b when used as an adsorbent fusion protein of the receptor TGF-b at a concentration of 1 μg / ml (Fig.27A) and 100 mg / ml (Fig.27B). The bold line indicates binding in the absence of the free TGF-b receptor. The dashed line indicates binding in the presence of the TGF-b receptor.

Drawings 28 through 31 show the resolution of retentate chromatography. On figa-28C shows the resolution of the proteins of the Hemophilus lysate using hydrophobic, cationic and Cu (II) adsorbents in the range of molecular weights from 0 kDa to 30 kDa. Each analyte retained is represented by a straight line, the height of which reflects the intensity of the analyte retained. On figa-29C shows the resolution of the proteins of the Hemophilus lysate using hydrophobic, cationic and Cu (II) adsorbents in the range of molecular weights from about 30 kDa to about 100 kDa. On Fig shows the combined resolution of Hemophilus proteins in the range from 0 kDa to 30 kDa on each of the three adsorbents. On Fig shows the combined resolution of Hemophilus proteins in the range from 20 kDa to 100 kDa on each of the three adsorbents.

On Fig shows the binding of the fused protein GST normal adsorbent.

FIGS. 33A-33B show the binding of a specific ligand to a fused GST receptor held on an adsorbent matrix (FIG. 33A), and the absence of ligand binding on a control matrix that does not contain a GST fusion receptor (FIG. 33B).

DETAILED DESCRIPTION OF THE INVENTION

1. DEFINITIONS

Unless otherwise indicated, all technical and scientific terms have the meaning commonly understood by those skilled in the art to which the invention belongs. In the following publications, a qualified person will find general definitions of many of the terms used in this description: Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY (2nd ed. 1994); THE CAMBRIDGE DICTIONARY OF SCIENCE AND TECHNOLOGY (Walker ed., 1988); THE GLOSSARY OF GENETICS, 5TH ED., R. Rieger et al., (Eds.), Springer Verlag (1991); and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY (1991). The following terms have the following meanings ascribed to them, unless otherwise indicated.

The term “analyte” means a component of a sample to be retained and detected. The term can mean either a single component or a set of components in a sample.

The term "Adsorbent" means any material capable of adsorbing analyte. The term “adsorbent” in the present context can mean both a single material (“monoplex adsorbent”) (i.e., a compound and a functional group) that the analyte is brought into contact with, and many different materials (“multiplex adsorbent”) in contact with which the sample is given. Adsorbent materials in a multiplex adsorbent are referred to as “adsorbent species”. For example, a predetermined localization on a substrate may contain many different types of adsorbent (i.e., anion exchange materials, metal chelators, or antibodies) having different binding characteristics.

The term “Adsorb” means detectable binding between the adsorbent and the analyte before or after washing with an eluant (selectivity threshold modifier).

The term "Substrate" means a solid phase to which an adsorbent is attached or held.

The term "binding Characterization" means the chemical and physical conditions that determine the analyte attraction of the adsorbent. Two adsorbents have different binding characteristics if, under the same elution conditions, the adsorbents bind the same analyte with a different degree of affinity. The binding characteristics include, for example, the degree of salt-dependent interaction, the degree of hydrophobic interaction, the degree

hydrophilic interaction, the degree of electrostatic interaction, etc.

The term “Binding Conditions” means the binding characteristics in which the analyte is processed.

The term "Eluant" means an agent, usually a solution, which is used to mediate the adsorption of analyte on an adsorbent. Eluants are also called "selectivity threshold modifiers."

The term “Elution Characteristic” means a property that determines the ability of a particular eluant (selectivity threshold modifier) to mediate adsorption between analyte and adsorbent. Two eluants have different elution characteristics if, when the analyte and the adsorbent are brought into contact, the degrees of affinity of the analyte to the adsorbent are different. Elution characteristics include, for example, pH, ionic strength, modification of the water structure, detergent strength, modification of hydrophobic interactions, etc.

The term "Elution Conditions" means the elution characteristics in which the analyte is processed.

The term “Selectivity Characterization” means a combination of an adsorbent having specific binding characteristics and an eluant having specific characteristics of an elution, wherein this combination determines the specificity with which the analyte will be retained by the adsorbent after washing with an eluant.

The term "Selectivity Conditions" means the selectivity characteristics in which the analyte is processed.

The term "base attraction" means the chemical or physico-chemical properties that cause the attraction of one molecule to another.

The term "Strength of Attraction" means the intensity of attraction of one molecule to another (also known as affinity).

The term "Allow", "resolution" or "resolution of the analyte" means the detection of at least one analyte in the sample. Resolution involves the detection of multiple analytes in a sample by separation and subsequent differential detection. Resolution does not necessarily require complete separation of the analyte from other analytes in the mixture.

The term "highly informative resolution" means the resolution of the analyte in a way that allows not only to detect the analyte, but also to evaluate at least one physicochemical property of the analyte, for example, molecular weight.

The term "Desorption spectrometry" means a method for detecting an analyte in which the analyte is exposed to energy that strips the analyte from the stationary phase into the gas phase and the desorbed analyte or its indistinguishable part is directly detected by the detector without an intermediate stage of analyte retention in the second stationary phase.

The term "Detect" means the establishment of the presence, absence or determination of the number of detected objects.

The term "Retention" (retention) means the adsorption of analyte by an adsorbent after washing with an eluant.

The term "Retention Data" means data indicative of the detection (further including a detectable mass) of an analyte retained under specific selectivity conditions.

The term "retention card" means a set of values characterizing the retention parameters of an analyte held under a variety of selective conditions.

The term “Recognition Profile” means a set of values characterizing the relative retention of the analyte under a variety of selective conditions.

The term "Complex" means analytes formed when two or more analytes are combined.

The term "fragment" means the products of chemical, enzymatic or physical cleavage of the analyte. Fragments may be neutral or ionic.

The term "Differential expression" means a detectable difference in the quantitative or qualitative presence of an analyte.

The term “biological sample” means a sample obtained from a virus, cell, tissue, organ or organism, including, without limitation, lysates or homogenates of a cell, tissue or organ, or samples of body fluids, such as blood, urine or cerebrospinal fluid.

The term "Organic Biomolecule" means an organic molecule of biological origin, i.e. steroids, amino acids, nucleotides, sugars, polypeptides, polynucleotides, complex carbohydrates or fats.

The term "Small Organic Molecule" means an organic molecule that is comparable in size to the organic molecules commonly used in pharmacology. The term excludes organic biopolymers (i.e. proteins, nucleic acids, etc.). Small organic molecules preferably have a size of up to 5000 Da, up to 2000 Da, or up to 1000 Da.

The term "Biopolymer" means a polymer of biological origin, i.e. polypeptides, polynucleotides, polysaccharides or polyslycerides (i.e. di- or triglycerides).

The term "Polypeptide" means a polymer consisting of amino acid residues, related natural structural variants, and synthetic, non-naturally occurring analogues. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. The term "protein" usually refers to large polypeptides. The term "peptide" usually refers to short polypeptides.

The term "Polynucleotide" means a polymer consisting of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include polynucleotides that include non-natural bases, nucleotides that provide a bond with other nucleotides that is different from the natural phosphodiester bond, or that contain bases joined by bonds other than phosphodiester bonds.

Thus, nucleotide analogs include, for example, but not limited to, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoroamidates, boranophosphates, methylphosphanates, chiral methyl phosphanates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs) and the like. Such polynucleotides can be synthesized, for example, using an automatic DNA synthesizer. The term "nucleic acid" usually refers to large polynucleotides. The term "oligonucleotide" usually refers to short polynucleotides not exceeding 50 nucleotides in length. It is understood that when the nucleotide sequence is a DNA sequence (i.e., A, T, G, C), this also applies to the RNA sequence (i.e., A, Y, G, C), where "Y" replaces the "T".

The term "detectable entity" or "label" means a compound detected by spectroscopic, photochemical, biochemical, immunochemical or chemical methods. For example, useful labels include ³² P, ³⁵ S, fluorescent dyes, electron-dense reagents, enzymes (e.g. those commonly used in ELISA), biotin-streptavidin, dioxigenin, haptens and proteins for which antisera or monoclonal are available antibodies, or nucleic acid molecules with a sequence complementary to the target. A detectable entity often provides a measurable signal, for example, a radioactive, chromogenic or fluorescent signal, which can be used to quantify a related detectable entity in a sample. A detectable entity can be integrated or attached to a primer or probe, either covalently or via ionic, van der Waals or hydrogen bonds, for example the incorporation of radioactive nucleotides or biotinylated nucleotides recognized by streptavidin. A detectable entity can be detected directly or indirectly. Indirect detection may involve linking a second directly or indirectly detectable entity. For example, a detectable entity may be a ligand for a binding partner, such as biotin, which is a streptavidin binding partner, or a nucleotide sequence that is a binding partner of a complementary sequence with which it can specifically hybridize. The binding partner itself can be directly detectable, for example, the antibody itself can be labeled with a fluorescent molecule. A binding partner may also be indirectly detectable, for example, a nucleic acid with a complementary nucleotide sequence may be part of a branched DNA molecule, which in turn is detectable by hybridization with other labeled nucleic acid molecules. (See, PDFahrlander and A. Klausner, Bio / Technology (1988) 6: 1165). Quantification of the signal can be carried out, for example, by scintillation counting, densitometry or flow cytometry.

“Many” means at least two.

"Cleanse" or "purification" means the removal of at least one impurity from the composition to be cleaned. Purification does not necessarily mean achieving 100% purity of the compound to be purified.

The term "Ligand" means a compound that specifically binds to a target molecule.

The term “receptor” means a compound that specifically binds to a ligand.

The term “Antibody” means a polypeptide ligand essentially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, that specifically binds to and is recognized by an epitope (i.e., antigen). Known immunoglobulin genes include the kappa and lambda light chain constant region of the genes, alpha, gamma, delta, epsilon and mu heavy chain of the constant region of the genes, and the myriad of variable regions of the immunoglobulin genes. Antibodies exist as intact immunoglobulins or as a series of well characterized fragments resulting from hydrolysis of various peptidases. These include Fab 'and F (ab)' ₂ fragments. The term "antibody", as used in the present context, also includes fragments of antibodies obtained by the modification of whole antibodies or synthesized de novoc using recombinant DNA methodology. These also include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, and humanized antibodies. An “Fc" fragment of an antibody means that part of the immunoglobulin heavy chain that contains one or more domains of the constant region of the heavy chain, CH ₁ , CH ₂ and CH ₃ , but does not contain the variable region of the heavy chain.

The ligand or receptor (i.e., antibody) "specifically binds to" or "specifically immunoreactive to" the analyte when the ligand or receptor functions in a binding reaction, indicating the presence of the analyte in the sample of heterogeneous compounds. Thus, under certain testing conditions (e.g. in an immunoassay), the ligand or receptor binds preferentially to a particular analyte and does not bind a small amount of other components of the sample. For example, a polynucleotide specifically binds under hybridization conditions to a polynucleotide analyte having a complementary sequence; the antibody specifically binds, under immunotest conditions, an antigen analyte carrying the epitope against which the antibody was obtained; the adsorbent specifically binds the analyte under appropriate elution conditions.

The term “Agent” means a chemical compound, a mixture of chemical compounds, a sample of an unspecified composition, a combinatorial order of small molecules, a biological macromolecule, a peptide library of bactriophages, a library of bactriophages carrying antibodies (ie, scFv), a peptide polysomal library, or an extract prepared from biological materials such as bacteria, plants, fungi, or animal cells and tissues. Acceptable techniques include screening libraries of recombinant antibodies in phages or similar vectors. See, Huse et al., (1989) Science 246: 1275-1281; and Ward et al., (1989) Nature 341: 544-546. The Huse protocol is more efficient when combined with phage library technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047.

The term "Recombinant polynucleotide" means a polynucleotide containing sequences that are not naturally linked. The amplified or “assembled” recombinant polynucleotide can be introduced into an acceptable vector, and the vector used to transform an acceptable host cell. A host cell containing a recombinant polynucleotide is referred to as a "recombinant host cell." Then, in a recombinant host cell, the gene is expressed to form a “recombinant polypeptide”. The recombinant polynucleotide can also perform a non-coding function (ie, serve as a promoter, start replication, ribosome binding site, etc.). Suitable unicellular hosts include any that are routinely used to express eukaryotic or mammalian polynucleotides, including, for example, prokaryotes, such as E. coli; eukaryotes, such as, for example, mushrooms and yeast; cultured cells, including insect cells (e.g., Sf9) and animal cells, such as CHO, R1.1, BW, LM, African monkey monkey kidney cells (e.g., COS 1, COS 7, BSC 1, BSC 40 and BMT 10), as well as cultured human cells.

The term “expression control sequence” means a nucleotide sequence in a polynucleotide that regulates the expression (transcription and / or translation) of an operably linked nucleotide sequence. "Operatively linked" means a functional relationship between two parts in which the activity of one part (i.e., the ability to regulate expression) leads to the realization of the activity of the second part (i.e., transcription of the sequence). Expression control sequences include, for example, but are not limited to, promoter sequences (e.g., inducible, suppressed, or constitutive), enhancers, transcription terminators, start codon (i.e. ATG), intron splicing and stop signals codons.

The term "Expression vector" means a vector containing a recombinant polynucleotide in which there are expression control sequences operatively associated with the nucleotide sequence to be expressed. The expression vector contains the necessary cis-acting elements for expression; other elements necessary for expression may be provided by the host cell or in vitro expression system. Expression vectors include all such vectors known in the art, for example, cosmids, plasmids (both naked and liposome-encapsulated) and viruses containing a recombinant polynucleotide.

The term “Coding” means the inherited property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes, while determining either a specific nucleotide sequence (ie rRNA, tRNA and mRNA ), or a specific amino acid sequence and biological properties due to them. Thus, a gene encodes a protein if transcription and translation of the mRNA synthesized on this gene leads to the formation of a protein in a cell or other biological system. As a coding chain, the nucleotide sequence of which is identical to the mRNA sequence and is usually given in databases, as well as a non-coding chain, used as a matrix for transcription, can be considered as encoding a protein or other product of a given gene or cDNA. Unless otherwise indicated, “nucleotide sequence encoding an amino acid sequence” means all nucleotide sequences that are degenerate versions of each other and which encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns.

The term "energy-absorbing molecule" means a molecule that absorbs energy from an energy source in desorption spectrometry, providing desorption of the analyte from the probe surface. The energy-absorbing molecules used in MALDI are often referred to as the “matrix”. Derivatives of cinnamic acid, cinapinic acid and dihydroxybenzoyl acid are often used as energy absorbing molecules for laser desorption of bioorganic molecules.

II RETENTANT CHROMATOGRAPHY

Retentate chromatography is a multidimensional resolution method for analytes in a sample. The method includes (1) selective adsorption of analytes of their sample on the substrate in many different adsorbent / eluant combinations (“selectivity conditions”) and (2) detection of adsorbed analyte retention by desorption spectrometry. Each selectivity condition provides one separation parameter or separation in one measurement, and adsorbed analytes are separated from non-adsorbed ones. Desorption spectrometry provides a second separation parameter, separating the adsorbed analytes from each other by mass. Since many different selectivity conditions are used in retentate chromatography, multidimensional separation is achieved. The relative adsorption of one or more analytes under two selectivity conditions can also be determined. This multidimensional separation provides both analyte resolution and its characterization.

In addition, analytes so separated remain on the retentate charts, which can be subjected to further analysis procedures, for example, to determine the structure of the analyte and / or its function. Also retained analytes themselves can serve as adsorbents, linking other analytes not carried onto the substrate. As a result, the present invention claims fast, multidimensional and highly informative resolution of analytes.

The method can be carried out in several forms or forms. According to one embodiment of the invention, the analyte is adsorbed on two different adsorbents in two physically different locations and each adsorbent is washed with the same eluant (selectivity threshold modifier). In another embodiment, the analyte is adsorbed on the same adsorbent in two physically different locations and washed with two different eluants. In another embodiment, the analyte is adsorbed on two different adsorbents in two physically different locations and washed with two different eluants. In another embodiment, the analyte is adsorbed onto the adsorbent, washed with the first eluant, and retention recorded; then the adsorbed analyte is washed with a second different eluant and subsequent retention is recorded.

A. Methods for performing retentate chromatography

1. Transfer of analyte to selective conditions

a. Substrate Preparation

When performing retentate chromatography, an analyte held by an adsorbent is exposed to an energy source on a substrate. The analyte-containing sample can be brought into contact with the adsorbent before or after the adsorbent is fixed to the substrate, which will serve to desorb the analyte. To ensure contact, the adsorbent can be presented in liquid or solid form (i.e., on a substrate or solid phase). A specific adsorbent may take the form of a solution, suspension, dispersion, water / oil emulsion, oil / water emulsion or microemulsion. When the adsorbent is in the form of a suspension, dispersion or microemulsion, an acceptable surfactant may also be included. In this embodiment, the sample can be brought into contact with the adsorbent by mixing the liquid sample with the liquid adsorbent. Alternatively, the sample can be deposited on a solid substrate, and contact is made by immersion, soaking or dipping of the solid sample supporting the sample in a liquid adsorbent. In addition, the sample can be brought into contact by spraying or rinsing the solid substrate with a liquid adsorbent. According to this embodiment, the various adsorbents may be in different containers.

In one embodiment, the adsorbent is applied to a substrate. The substrate can be any material that is able to bind and hold. Typically, the substrate may be glass; ceramics;

electrically conductive polymers (i.e. carbonated PEEK); TEFLON ^B coated materials; organic polymers, natural biopolymers; metals (i.e. nickel, brass, steel or aluminum); films; porous and non-porous beads and cross-linked polymers (i.e., agarose, cellulose or dextran); other insoluble polymers or combinations thereof.

According to one embodiment of the invention, the substrate is in the form of a probe or sample inserted into a desorption detector. For example, as can be seen from figure 1, the substrate may be in the form of strips. The adsorbent can be attached to the substrate in the form of a linear order of spots, each of which is brought into contact with the analyte. Several bands can be combined so that many adsorbents form a matrix with individual spots in each particular row. The substrate may also be in the form of a plate, consisting of horizontal and vertical rows of adsorbents, forming the correct geometric order, such as a square, rectangle or circle.

Samples can be obtained as follows. The substrate may be any solid material, for example, stainless steel, aluminum or silicon gratings. The metal substrate is then coated with a material that allows surface derivatization. For example, a metal surface can be coated with silicon oxide, titanium oxide or gold.

Then the surface is derivatized with a bifunctional linker, at one end the linker contains a functional group that covalently binds to a functional group on the surface. This functional group may be an inorganic oxide or sulfhydryl group in the case of gold. At the other end, the linker typically has an amino group. Useful bifunctional linkers include aminopropyl triethoxysilane or aminoethyl disulfide.

After the linkers are attached to the surface, they are further derivatized by groups that function as adsorbent. Typically, the adsorbent is applied at predetermined locations on the probe. In one case, the probe spots are about 3 mm in diameter and assembled in the form of a rectangular matrix. Adsorbents can themselves be part of bifunctional molecules containing a group that reacts with an available amino group, and a functional group that acts as an adsorbent. Functional groups include, for example, normal phase (silica), reverse phase (C ₁₈ aliphatic hydrocarbon), quaternary amine and sulfonate. In addition, the surface can be further derivatized with other bifunctional molecules, such as carbodiimide and N-hydroxysuccinimide, to obtain a preactivated preform. Such preforms can be derivatized with bioorganic adsorbents (i.e., nucleic acids, antibodies, and other protein ligands). Biopolymers can bind to the functional groups of the preforms via amine residues or sulfhydryl residues.

According to one embodiment of the invention, the adsorbents are attached to cross-linked polymers (i.e., films) that are associated with the failure of the probe through accessible functional groups. Such polymers include, for example, cellulose, dextran, carboxymethyl dextran, polyacrylamide and mixtures thereof. Probes with attached adsorbents are ready for use.

According to another embodiment of the invention, the adsorbent is attached to the first substrate to obtain a solid phase, for example, to polymer or glass beads, which are then placed on a second substrate, which serves to present a sample of the desorption energy of the desorption detector. For example, the second substrate may be in the form of a plate with rows of cells in predetermined locations. The cells can serve as containers for a first substrate derivatized with an adsorbent, for example polymer beads, derivatized with an adsorbent. One of the advantages of this option is that the analyte can be adsorbed by the first substrate in one physical context and transferred to a representative substrate for analysis by desorption spectrometry.

Typically, the substrate is adapted for use with the used detectors, which in accordance with the present invention are used to detect analyte bound to and retained by the adsorbent. According to one embodiment of the invention, the substrate is inserted into a desorption detector, where the energy source can be directed to the spot and cause desorption of the analyte. The substrate can be adapted for mounting on a vertically and / or horizontally movable carrier, which vertically and / or horizontally moves the substrate in successive positions, substituting the predetermined adsorbent localization for the energy source to detect analytes bound to them. The substrate may take the form of a conventional mass spectrometer probe.

Strips, plates or substrate probes can be obtained by conventional methods. Then, the adsorbent must be directly or indirectly bound, combined, or located on the substrate before being brought into contact with a sample containing the analyte. The adsorbent can be directly or indirectly connected to the substrate by any suitable method of attachment or immobilization. For example, the adsorbent can be directly attached to the substrate by derivatization of the substrate with the adsorbent by the formation of covalent or non-covalent bonds.

The attachment of the adsorbent to the substrate can be carried out by various mechanisms. The substrate can be derivatized with a fully prepared adsorbent molecule by attaching a previously prepared adsorbent molecule to the substrate. Alternatively, the adsorbent can be formed on the substrate by attaching precursor molecules to the substrate, followed by the addition of additional precursor molecules to the growing chain attached to the substrate of the first precursor molecule. This mechanism of constructing an adsorbent on a substrate is especially useful when the adsorbent is a polymer, in particular a biopolymer such as a DNA or RNA molecule. A biopolymer adsorbent can be prepared by sequentially adding bases to the first base attached to the substrate using oligonucleotide chip technology techniques. See, e.g., U.S. Patent No. 5,445,934 (Fodor et al.).

As can be seen from figure 2, only two or as many as 10, 100, 1000, 10000 or more adsorbents can be associated with one substrate. The size of the adsorbent site may vary depending on the method of setting up the experiment and its goals. However, it should not be larger than the diameter of the energy source used (i.e., the diameter of the laser spot). Stains may contain the same or different adsorbents. In some cases, it is preferable to obtain the same adsorbent in multiple locations on the substrate in order to evaluate the effects of many different eluants, and so that the bound analyte can be stored for later use as a reference or for subsequent processing. Obtaining a substrate with many different adsorbents, you can use many of the characteristics of the binding provided by the combination of different adsorbents in relation to one sample, and therefore to bind and detect a wider variety of different analytes. The use of many different adsorbents on a substrate to evaluate a single sample is essentially the equivalent of competitively performed multiple chromatographic experiments, each of which requires a separate chromatographic column. An advantage of the present invention is that its implementation requires only one chromatographic system.

When the substrate contains many adsorbents, it is especially useful to obtain adsorbents in predetermined locations. Obtaining adsorbents in predetermined localizations, it is possible to wash the adsorbent in the first predetermined localization with the first eluant and wash the adsorbent in the second predetermined localization with the second eluant. Thus, the binding characteristics of a single adsorbent in relation to a given analyte can be evaluated in the presence of multiple eluants, each of which selectively modifies the binding characteristics of the adsorbent in its own way.

Predefined localizations can be arranged in order, with the correct orders being preferred, such as rows, rectangular matrices, or regular curves, such as circles. Similarly, when the substrate contains many adsorbents, one particular eluant can be evaluated for each different adsorbent to obtain binding characteristics of the adsorbent in the presence of eluant. You can also evaluate the binding characteristics of various adsorbents in the presence of various eluants.

(1) Rising or gradient adsorbing surfaces

You can get a series of adsorbents with different binding characteristics, synthesizing many different polymer adsorbents on the substrate. Various polymeric adsorbents can be obtained by attaching a precursor molecule to the substrate, initializing the polymerization reaction and terminating the polymerization reaction at different stages of completion for each adsorbent. The terminal functional groups of polymers can be chemically derivatized to various degrees by various affinity reagents (e.g., -NH ₃ , or COO ^- ). When terminating a polymerization or derivatization reaction, adsorbents with different degrees of polymerization or derivatization are obtained. Different degrees of polymerization or derivatization provide different binding characteristics for each individual polymer adsorbent. This embodiment of the invention is particularly applicable to the preparation of many different biopolymer adsorbents on a substrate.

If desired, the polymerization reaction can be carried out in the reaction vessel, and not on the substrate itself. For example, polymeric adsorbents with different binding characteristics can be obtained by extracting an aliquot of the product from the reaction vessel during the polymerization / derivatization reaction. Aliquots taken at different time points during the polymerization / derivatization reaction will have varying degrees of polymerization / derivatization to produce many different adsorbents. Different aliquots of the product can then be used as adsorbents with different binding characteristics. Alternatively, many different adsorbents can be obtained by sequentially repeating the step of terminating the reaction, selecting an aliquot of the product and re-starting the polymerization / derivatization reaction. The products obtained at each termination point will have a different degree of polymerization / derivatization and as a result will produce many adsorbents with different binding characteristics.

According to one embodiment of the invention, the substrate is obtained in the form of a strip or plate coated with an adsorbent, in which one or more binding characteristics vary in a one- or two-dimensional gradient. For example, a strip is obtained with an adsorbent that is weakly hydrophobic at one end and highly hydrophobic at the other. Or get a plate that is weakly hydrophobic and anionic in one corner and strongly hydrophobic and anionic in a diagonally opposite corner. Such adsorption gradients are useful in the qualitative analysis of the analyte. Adsorption gradients can be obtained by controlled sputtering or pouring of material onto the surface for a time that would provide an increasing completion of the reaction with the formation of a gradient. This process can be repeated at right angles to obtain rectangular gradients of similar or different adsorbents with different binding characteristics.

The analyte-containing sample can be brought into contact with the adsorbent before or after the adsorbent is located on the substrate in a convenient manner that provides binding between the analyte and the adsorbent. The adsorbent can simply be mixed with the sample. The sample can be brought into contact with the adsorbent by dipping or immersing the substrate in the sample, spraying the sample over the substrate, washing the substrate with the sample, or by directly forming the sample or analyte in contact with the adsorbent. In addition, the sample can be brought into contact with the adsorbent by solubilization of the sample or by mixing the sample with eluant and bringing into contact with the adsorbent a solution of the eluant with the sample using any of the following methods (e.g., dipping, dipping, spraying or washing).

b. Bringing the analyte into contact with the adsorbent

Processing the sample with the eluant before binding the analyte with an adsorbent has a modifying effect on the selectivity of the adsorbent, while the contact of the sample with the adsorbent occurs simultaneously. The components of the sample that will bind to the adsorbent and therefore be retained include only those components that will bind to the adsorbent in the presence of a particular eluant, and not all components that will bind to the adsorbent in the absence of elution characteristics that modify the selectivity of the adsorbent.

The sample must be brought into contact with the adsorbent for such a period of time that is sufficient to bind the analyte to the adsorbent. Typically, the sample is brought into contact with the adsorbent for a period of time from 30 seconds to 12 hours. Preferably, the sample is brought into contact with the adsorbent for a period of time from 30 seconds to 15 minutes.

The temperature at which the sample is brought into contact with the adsorbent depends on the particular sample and the selected adsorbents. Typically, the sample is brought into contact with the adsorbent at normal temperature and pressure, however, for some samples, you may need a modified temperature (usually from 4 ° C to 37 ° C) and pressure, which are quite easily determined by a qualified specialist.

Another advantage of the present invention over conventional detection methods is that it enables numerous different experiments with a very small amount of sample. Typically, the sample contains from several atomomoles to 100 picomoles of analyte in a volume of 1 μl to 500 μl, which is sufficient for binding to the adsorbent. The analyte can be saved for subsequent experiments after binding by the adsorbent, since any localization of the adsorbent that has not been subjected to the steps of desorption and detection of all retained analyte will retain the analyte. Accordingly, in cases where a sample of a very small volume is available for analysis, the present invention has the advantage and makes it possible to conduct many experiments in various adsorbents and / or eluants at different times without washing the sample.

in. Washing the adsorbent with eluants

After the sample is brought into contact with the analyte, which ensures the binding of the analyte to the adsorbent, the adsorbent is washed with eluant. Typically, in a multivariate analysis, the adsorbent in each spot is washed with at least the first and second different eluents. Washing with eluants modifies the analyte population retained by the specific adsorbent. The combination of adsorbent binding characteristics and eluant elution characteristics provides selectivity conditions holding analytes on the adsorbent after washing. Thus, the washing step selectively removes the components of the sample from the adsorbent.

The washing step can be carried out using a variety of methods. For example, as shown above, the sample can be solubilized or mixed with the first eluant before bringing the sample into contact with the adsorbent. Processing the sample with the first eluant before or simultaneously with contacting it with the adsorbent essentially has the same effect as binding the analyte to the adsorbent, followed by washing the adsorbent with the first eluant. After the mixed solution is applied to the adsorbent, the adsorbent can be washed with a second or subsequent eluant.

Washing of the adsorbent with analyte attached to it is achieved by dipping, immersing the substrate in eluant; or rinsing, spraying, or pouring eluant onto a substrate. The introduction of eluant into the spots of a small diameter affinity reagent is best achieved by the microfluidization process.

When the analyte is attached to the adsorbent in only one location, and many different eluants are used in the washing steps, information on the selectivity of the adsorbent individually in the presence of each eluant can be obtained. An analyte attached to the adsorbent in one localization can be determined after each washing with the eluant, sequentially washing with the first eluant, desorption and detection of the retained analyte, washing with the second eluant, desorption and detection of the retained analyte. Desorption and detection following the washing steps can be sequentially repeated with many different eluants on the same adsorbent. Thus, the adsorbent with an analyte attached to it in a single localization can be cross-checked with many different eluants to obtain information about the analytes retained after each individual washing.

The methods described below are also applicable when the adsorbents are in many predetermined locations, regardless of whether it is one adsorbent or different. However, when the analyte is bound to the same adsorbent or to different adsorbents in multiple locations, the washing step can alternatively be carried out using a more systematic and efficient parallel processing approach. Namely, the washing step is carried out by washing the adsorbent with the eluant in the first localization, then washing the second adsorbent with the eluant, then desorbing and detecting the analyte retained by the first adsorbent, and then desorbing and detecting the analyte retained by the second adsorbent. In other words, all adsorbents are washed with eluant, and then the analyte retained by each of them is desorbed and detected for each adsorbent location. Optionally, after detection in each localization of the adsorbent, a second stage of washing each localization of the adsorbent can be carried out, followed by a second stage of desorption and detection. The washing steps of all adsorbent locations, followed by desorption and detection at each adsorbent location, can be repeated for many different eluants. In this way, the entire matrix can be used to effectively determine the nature of the analytes in the sample. This method is applicable regardless of whether all adsorbent locations are washed with one eluant in the first washing step or many adsorbents are washed with many different eluants in the first washing step.

2. Detection

Analytes retained by the adsorbent after washing are adsorbed on the substrate. The analytes held on the substrate are detected by desorption spectrometry: the analyte is desorbed from the adsorbent and the desorbed analytes are directly determined.

a. Desorption Methods

Desorption of the analyte from the adsorbent involves treating the analyte with an appropriate energy source. This usually means processing the analyte with radiation or energy particles. For example, the energy may be in the form of light energy, for example, laser energy (i.e., a UV laser), or energy from a flash lamp. Alternatively, energy can be in the form of a stream of fast atoms. Heat can also be used to induce desorption.

Methods for desorption and / or ionization of analytes for direct analysis are well known in the art. One such method is called matrix laser desorption / ionization or MALDI. During MALDI, the analyte solution is mixed with the matrix solution and the mixture is allowed to crystallize on the surface of an inert probe; analyte retention in crystals creates the possibility of desorption. The matrix is chosen so that it absorbs laser energy and directly transfers it to the analyte, making desorption and ionization possible. Typically, the matrix absorbs in the UV region. MALDI for large proteins is described, for example, in US Pat. No. 5,118,937 (Hillenkamp et al.) And US Pat. No. 5,045,694 (Beavis and Chait).

Surface-enhanced laser desorption / ionization, or SELDI, has a significant advantage over MALDI in terms of specificity, selectivity and sensitivity. SELDI is described in US Pat. No. 5,719,060 (Hutchens and Yip). SELDI is a solid phase desorption method whereby an analyte is presented to a surface energy source that enhances analyte retention and / or desorption. Conversely, MALDI is a liquid phase method and the analyte is mixed in a liquid material that crystallizes around the analyte.

One variant of SELDI, called SEAC (Surface-Enhanced Affinity Capture), involves the presentation of the analyte to a source of desorption energy in combination with an affinity restraint (i.e., adsorbent). It has been shown that when the analyte is adsorbed in this way, it can be presented to a source of desorbing energy with great potential for achieving desorption of the target analyte. To facilitate desorption, an energy-absorbing material may be added to the probe. The probe is then presented to an energy source for desorption of the analyte.

Another version of SELDI, called SEND (Surface-Enhanced Neat Desorption), involves the use of a layer of energy-absorbing material on which the analyte is placed. The surface of the substrate contains a layer of energy-absorbing molecules chemically bonded to the surface and / or substantially free of crystals. Then a pure (undiluted) analyte is applied to the surface of the layer without significant mixing with it. Molecules absorbing energy, like the matrix, absorb desorbing energy, which leads to desorption of the analyte. This improvement is significant because analytes can be presented to the energy source in a simpler and more uniform way, since the contribution of mixed solutions and random crystallization are excluded. This gives more uniform and predictable results and makes it possible to automate the process. The energy absorbing material may be a classic matrix material, or it may be a matrix material whose pH is neutralized or in the main range. Energy absorbing molecules can be attached to the probe covalently or non-covalently.

Another version of SELDI called SEPAR (Surface-Enhanced Photolabile Attachment and Release) uses photolabile attachment molecules. A photolabile attachment molecule is a divalent molecule in which one site is covalently linked to a solid phase, such as a flat surface of the probe, or to another solid phase, such as a beads, and the second site can be covalently linked to an affinity reagent or analyte. The photolabile attachment molecule, when bound to the surface and analyte, also contains a photolabile bond that can release the affinity reagent or analyte under the action of light. The photolabile bond can be located both in the attachment molecule and in the attachment site of the analyte (or affinity reagent) or the surface of the probe.

b. Direct analyte detection method

Desorbed analyte can be detected in various ways. When the analyte is ionized during the desorption process, as is the case with laser desorption-ionization mass spectrometry, the detector may be an ion detector. Mass spectrometers can usually determine the flight time of desorbed ions. This information gives an idea of the mass of ions. However, for the resolution and detection of desorbed ions, there is no need to determine their mass: the fact that ionized analytes hit the detector at different times ensures their resolution and detection.

Alternatively, the analyte may be labeled, for example, with a fluorescent or radioactive label. In these cases, the detector may be a fluorescence or radioactivity detector.

Many detection methods can be used sequentially to fully examine the analyte components or the function associated with the analyte held in each matrix location.

in. Desorption Detectors

Desorption detectors make it possible to desorb analyte from an adsorbent, as well as the possibility of direct detection of desorbed analyte. That is, the desorption detector detects the desorbed analyte without an intermediate step of retaining the analyte in another solid phase. Detection of an analyte usually involves the determination of signal strength. It, in turn, reflects the amount of analyte adsorbed by the adsorbent.

In addition to these two elements, the desorption detector may also include other elements. One of these elements makes it possible to accelerate the desorbed analyte towards the detector. Another element makes it possible to determine the flight time of the analyte from the moment of desorption to the moment of detection by the detector.

A preferred desorption detector is a laser desorption ionization mass spectrometer, well known in the art. The mass spectrometer has a port into which the probe is inserted, i.e. substrate containing adsorbed analytes. Desorption is carried out due to the energy transferred to the analyte, in particular laser energy. The device provides surface movement so that any spot on the matrix gets under the laser beam. Laser processing of the analyte leads to desorption of the intact analyte into the sleeve (flight tube) and its ionization. A vacuum is usually created in the sleeve. Charged plates located in a certain section of the sleeve create an electric potential that accelerates the ionized analyte in the direction of the detector. The clock measures flight time, and the electronic system determines the speed of the analyte and converts it to mass. As is obvious to any skilled person, any of these elements can be combined with the other elements described when assembling desorption detectors that use various approaches for desorption, acceleration, detection, time measurement, etc.

B. Conditions of selectivity

One of the advantages of the present invention is the ability to process analytes in a variety of different conditions of binding and elution, thereby obtaining increased resolution of the analytes and information about them in the form of a recognition profile. As in the case of conventional chromatographic methods, the ability of the adsorbent to retain the analyte directly depends on the attraction or affinity of the analyte to the adsorbent compared with the attraction or affinity of the analyte to the eluant or eluant to the adsorbent. Some components of the sample may not have affinity for the adsorbent and therefore will not be bound by the adsorbent when the sample is brought into contact with this adsorbent. Due to their inability to bind to the adsorbent, these components will be immediately separated from the analyte to be resolved. However, depending on the nature of the sample and the particular adsorbent used, various components may be initially attached to the adsorbent.

1. Adsorbents

Adsorbents are materials that bind analytes. In retentate chromatography, many adsorbents can be used. Different adsorbents may have very different binding characteristics, moderately different binding characteristics, or slightly different binding characteristics. Adsorbents having very different binding characteristics usually differ in affinity fundamentals or in the manner of interaction. The basis of affinity is usually a function of chemical or biological molecular recognition. The fundamentals of affinity between an adsorbent and an analyte include, for example, (1) salt-potentiated interactions, i.e. hydrophobic interactions, thiophilic interactions and interactions with immobilized dyes; (2) interactions based on hydrogen bonds and / or Van der Waals forces, as well as interactions based on charge transfer, as in the case of hydrophilic interactions; (3) electrostatic interactions, such as interactions of ionic charges, in particular interactions of positive and negative ions; (4) the ability of the analyte to form coordinate covalent bonds (i.e., the formation of a coordinate complex) with a metal ion in the adsorbent; (5) binding to the active site of the enzyme; (6) reversible covalent interactions, for example, interactions based on disulfide substitution; (7) glycoprotein interactions; (8) biospecific interactions; or (9) a combination of two or more of these types of interactions. An adsorbent having two or more affinity bases is called an “mixed functionality” adsorbent.

a. Salt Potentiated Adsorbents

Adsorbents that are applicable for studying salt potentiated interactions include hydrophobic interaction adsorbents. Examples of hydrophobic interaction adsorbents are matrices with aliphatic hydrocarbons, in particular C ₁ -C ₁₈ aliphatic hydrocarbons, and matrices with aromatic hydrocarbon functional groups, such as phenyl groups. Hydrophobic interaction adsorbents bind analytes that exhibit uncharged amino acid residues, in particular amino acid residues such as non-polar, aromatic and hydrophobic amino acid residues such as phenylalanine and tryptophan. Specific examples of analytes that will bind to the adsorbent hydrophobic interaction are lysozyme and DNA. Without striving to be limited to any particular theory, it is believed that DNA binds to adsorbents of hydrophobic interaction through aromatic nucleotides, in particular purine and pyrimidine groups.

Another type of adsorbents that are useful for studying salt potentiated interactions include thiophilic interaction adsorbents, such as, for example, T-GEL ^B , which is one of the types of thiophilic interaction adsorbents commercially available from Pierce, Rockford, Illinois. Thiophilic interaction adsorbents bind, for example, immunologicins such as IgG. The mechanism of interaction between IgG and T-GEL ^B is not fully established, but, apparently, the exposed tryptophan residues play a role in it.

The third type of adsorbents, in which salt-promoted ionic interactions and also hydrophobic interactions are involved, are adsorbents of interaction with an immobilized dye. Immobilized dye interaction adsorbents include immobilized dye matrices such as CIBACRON ™ Blue, manufactured by Pharmacia Biotech, Piscataway, New Jersey. Adsorbents of interaction with immobilized dye bind proteins and DNA in general. One specific example of a protein that binds to adsorbents of interaction with an immobilized dye is bovine serum albumin (BSA).

b. Adsorbents of hydrophilic interaction

Adsorbents that are applicable to the study of hydrogen bonds or Vanderwaals forces as the basis for hydrophilic interaction include surfaces containing a normal phase adsorbent such as silicon oxide (i.e. glass). The normal phase or silicon oxide surface acts as a functional group. In addition, adsorbents represented by surfaces modified by hydrophilic polymers such as polyethylene glycol, dextran, agarose or cellulose can also act as adsorbents for hydrophilic interaction. Most proteins will be bound by adsorbents of hydrophilic interaction due to the presence of groups or combinations of amino acid residues (i.e., hydrophilic amino acid residues) that bind by hydrophilic interaction involving hydrogen bonds or Vandervaals forces. Examples of proteins that will be bound by adsorbents of hydrophilic interaction are myoglobin, insulin and cytochrome C.

In general, proteins with a high proportion of polar or charged amino acids will be retained on the hydrophilic surface. Alternatively, glycoproteins with exposed hydrophilic grappa sugars also have high affinity for hydrophilic adsorbents.

in. ESD adsorbents

Adsorbents suitable for the study of electrostatic or ion-charged interactions include such anionic adsorbents as, for example, matrices with sulfate anions (i.e., SO ₃ ^- ), matrices with carboxylate anions (i.e. COO ^- ) or phosphate anions (OPO ₃ ^- ). Matrices with sulfate anions are constantly negatively charged. However, matrices with carboxylate anions are negatively charged only at a pH above pKa. At pH below pKa, the matrices have a substantially neutral charge. Acceptable anionic adsorbents also include matrices with a combination of sulfate, carboxylate and phosphate anions. The combination provides such a negative charge intensity that can constantly vary as a function of pH. These adsorbents attract and bind positively charged proteins and macromolecules, such as, for example, ribonuclease and lactoferrin, without striving to be limited by any particular theory, it is believed that the electrostatic interaction between the adsorbent and positively charged amino acid residues, including lysine residues, arginine residues and histidyl residues responsible for the binding interaction.

Other adsorbents useful for studying electrostatic or ion-charged interactions are cationic adsorbents. Specific examples of cationic adsorbents are matrices of secondary, tertiary and quaternary amines. Quaternary amines are constantly positively charged. However, the charge of secondary and quaternary amines is pH dependent. At a pH below pKa, the secondary and quaternary amines are positively charged, and at a pH above pKa they are negatively charged. Acceptable cationic adsorbents also include cationic adsorbents, which are matrices with a combination of various secondary, tertiary and quaternary amines. The combination provides such a positive charge intensity that can constantly vary as a function of pH. Adsorbents of cationic interaction bind anionic sites on molecules, which include, for example, proteins with exposed amino acid residues, such as residues of aspartic acid and glutamic acid.

In the case of adsorbents of ionic interaction (both anionic and cationic), it is often required to obtain mixed adsorbents containing both anions and cations. Such adsorbents provide a constant buffer capacity as a function of pH. The constant buffer capacity makes it possible to process analyte combinations with eluants having different buffer components, especially in the pH range from 2 to 11. This leads to the formation of a local pH environment on the adsorbent, which is determined by immobilized titratable proton-exchange groups. Such systems are equivalent to the solid phase separation method known as chromatofocusing. Isoforms of follicle-stimulating hormone, which differ mainly in the content of charged carbohydrates, are separated on a chromatofocusing adsorbent.

Other adsorbents useful for studying electrostatic interactions include dipole-dipole interaction adsorbents, in which the interaction is electrostatic, but does not involve a formal charge or titratable donor or acceptor.

d. Adsorbents coordinate covalent interaction

Adsorbents suitable for studying the ability to form coordinate covalent bonds with metal ions include matrices that carry, for example, ions of divalent and trivalent metals. Matrices with immobilized metal ion chelators are prepared on the basis of immobilized organic synthetic molecules that have one or more electron-donor groups that form the basis for coordinate covalent interactions with transition metal ions. Primary electron-donor groups that function as immobilized metal ion chelators include oxygen, nitrogen, and sulfur. Metal ions bind to immobilized metal ion chelators, which leads to the formation of a complex of metal ions, which still has a certain number of sites for interaction with electron-donor groups of the analyte. Acceptable metal ions generally include transition metal ions such as copper, nickel, cobalt, zinc, iron, as well as other metal ions, such as aluminum and calcium. Without striving to be limited to any particular theory, it is believed that metal ions interact selectively with specific amino acid residues of peptides, proteins, or with nucleic acids. Typically, the amino acid residues involved in such interactions include histidine residues, tyrosine residues, tryptophan residues, cysteine residues and amino acid residues containing oxygen groups such as aspartic acid and glutamic acid. For example, immobilized iron ions interact with phosphoserine, phosphotyrosine, and phosphotreonine residues in proteins. Depending on the immobilized metal ion, only those proteins that have a sufficient local density of previous amino acid residues will be retained by the adsorbent. In some cases, the interaction between metal ions and proteins is so strong that, using conventional methods, it is not possible to liberate the protein from the complex of human b-casein, which is highly phosphorylated, binds very strongly to immobilized Fe (III). Recombinant proteins bearing the label of 6-histidine bind very strongly to immobilized Cu (II) and Ni (II).

D. Adsorbents based on interaction with the active site of the enzyme

Adsorbents useful for studying interactions with the active site of an enzyme include proteases (such as trypsin), phosphatases, kinases, and nucleases. In this case, the interaction is a sequence-specific interaction of the enzyme binding site on the analyte (usually a biopolymer) with the catalytic binding site of the enzyme. The binding sites of an enzyme of this type include, for example, active trypsin sites that interact with proteins and peptides having lysine-lysine and lysine-arginine fragments in their sequences. More specifically, a soybean trypsin inhibitor interacts and binds to an adsorbent with immobilized trypsin. Alternatively, serine proteases are selectively retained on an adsorbent with immobilized L-arginine.

e. Adsorbents of reversible covalent interaction

Adsorbents applicable to the study of reversible covalent interaction include disulfide interaction adsorbents. Disulfide adsorbents include adsorbents with immobilized sulfhydryl groups, i.e. mercapoethanol or immobilized dithiothreitol. The interaction is based on the formation of covalent disulfide bonds between the cysteine residues on the analyte after being contacted with an adsorbent and a solvent. Such adsorbents bind proteins or peptides having cysteine residues, as well as nucleic acids that contain bases modified with reduced sulfur.

g. Glycoprotein Interactions

Glycoprotein interaction adsorbents include adsorbents with immobilized pectins (i.e., oligosaccharide-bearing proteins), exemplified by CONCANAVALIN ™ manufactured by Pharmacia Biotech, Piscataway, New Jersey. Such adsorbents function based on an interaction in which molecular recognition of the hydrocarbon portion of the macromolecules is involved. Examples of analytes that interact and bind with glycoprotein interaction adsorbents are glycoproteins, in particular glycoproteins rich in histidine, whole cells and isolated subcellular fractions.

h. Adsorbents of biospecific interaction

Adsorbents useful for studying biospecific interactions are commonly referred to as “biospecific affinity adsorbents”. Adsorption is considered biospecific if it is specific and affinity (equilibrium dissociation constant, Kd) is at least 10 ^-3 M (e.g. 10 ^-5 M, 10 ^-7 M, 10 ^-9 M). Examples of biospecific affinity adsorbents are any adsorbents that specifically interact with and bind to a particular biomolecule. Adsorbents of biospecific affinity include, for example, immobilized antibodies that bind antigens; immobilized DNAs that bind DNA binding proteins, DNA and RNA; immobilized substrates or inhibitors that bind proteins and enzymes; immobilized drugs that bind the drug - binding proteins; immobilized ligands that bind receptors; immobilized receptors that bind ligands; immobilized RNAs that bind DNA and RNA binding proteins; immobilized avidin or streptavidin that bind biotin and biotinylated molecules; immobilized phospholipid membranes and vesicles that bind lipid binding proteins. Enzymes serve as useful adsorbents that can modify adsorbed analyte. Cells can also be useful adsorbents. A complex of binding characteristics is presented on their surfaces. Cell adsorption is useful, for example, to identify ligands or signaling molecules that bind to surface receptors. Viruses or phages can also be useful adsorbents. Viruses often carry ligands to cell surface receptors (e.g. gp l20 for CD4). Also in the form of a phage library, phage surface proteins act as agents for testing binding to targets. Adsorbents for biospecific interactions are based on known specific interactions similar to those listed above.

According to one embodiment of the invention, the biospecific adsorbent may also contain an additional or “helper” molecule that is not directly involved in the binding of the target analyte.

g. The degree of binding specificity

By bringing into contact with adsorbents having various interaction methods, the components contained in the sample can mainly be separated based on their interaction with various adsorbents. Thus, the attraction of the analyte with adsorbents having various interaction methods provides the first separation parameter. For example, bringing a sample containing an analyte into contact with a first adsorbent whose affinity is based on hydrophobicity and a second adsorbent whose affinity is based on an ionic charge, it is possible to isolate those analytes that bind to a hydrophobic adsorbent from the sample and to separate those analytes that bind to an adsorbent having a specific ionic charge.

Adsorbents having different affinity bases provide analyte resolution with a low degree of specificity, since the adsorbent will bind not only the analyte, but also any other components of the sample having the same affinity basis. For example, a hydrophobic adsorbent will bind not only a hydrophobic analyte, but also any other hydrophobic components of the sample; a negatively charged adsorbent will bind not only a positively charged analyte, but also any other positively charged components of the sample; etc.

The resolution of analytes based on the affinity of the analyte to the adsorbent can be enhanced by applying binding characteristics relative to intermediate specificity or altered attraction strength. Separation of the analyte based on the binding characteristics of intermediate specificity can be achieved, for example, using adsorbents of mixed functionality. After the resolution of the analyte is completed with relatively low specificity, the binding characteristic for which the analyte of interest is indicated can be used in combination with various other binding or elution characteristics to remove a larger number of undesirable components and thereby achieve a higher resolution. analyte.

For example, if the analyte is bound by hydrophobic adsorbents, then it can be further resolved from other hydrophobic components of the sample, obtaining an adsorbent of mixed functionality, in which hydrophobic interaction serves as one basis for affinity, but there is also an excellent, different basis for affinity. The adsorbent of mixed functionality may have a hydrophobic interaction and an interaction based on negatively charged ions, binding hydrophobic analytes that are positively charged. Alternatively, an adsorbent of mixed functionality may have a hydrophobic interaction and the ability to form coordinate covalent bonds with metal ions, linking hydrophobic analytes that have the ability to form coordinate complexes with metal ions on the adsorbent. Other examples of adsorbents having binding characteristics of intermediate specificity will be apparent to those skilled in the art, taking into account the descriptions and examples given in this application.

The separation of analytes based on binding characteristics of intermediate specificity can be further enhanced by using the binding characteristics of relatively high specificity. The binding characteristics of relatively high specificity can be used using different adsorbents with the same affinity basis, but with different affinity strengths. In other words, while the basis of affinity remains the same, finer resolution of the analyte from other components of the sample can be achieved by using adsorbents having different degrees of affinity for the analyte.

For example, an analyte that binds to an adsorbent based on the acidic nature of the analyte can be separated from other acidic components of the sample by using adsorbents with affinity for analytes in specific acidic pH ranges. Thus, the analyte can be resolved using one adsorbent that extracts the components of the sample in the range of pH 1-2, another adsorbent that extracts the components of the sample in the range of pH 3-4, and a third adsorbent that extracts the components of the sample in the range of pH 5-6. Thus, an analyte having a specific affinity for an adsorbent that binds an analyte in the pH range of 5-6 will be separated from sample components having a pH in the pH range of 1-4. Adsorbents of increasing specificity can be used to reduce the range of affinity, i.e., differences in the binding characteristics of adsorbents based on the same nature of affinity.

The primary analyte adsorbed on the primary adsorbent may itself have adsorbent properties. In this case, the primary analyte adsorbed on the substrate may become a secondary adsorbent to isolate secondary analytes. In turn, the retained secondary analyte can function as a quaternary adsorbent to isolate the quaternary analytes from the sample. This process can be repeated several times.

2. Eluants

Eluants or washings selectively modify the adsorption threshold between the analyte and the adsorbent. The ability of the eluant to desorb and elute the bound analyte is a function of its elution characteristics. Different eluants can have very different elution characteristics, moderately different elution characteristics, or slightly different elution characteristics.

Typically, the eluant is brought into contact with the adsorbent at a temperature of from 0 ° C to 100 ° C, preferably in the range from 4 ° C to 37 ° C. However, for some eluants, a different temperature may be required that is readily determined by a qualified person.

As with adsorbents, eluants with very different elution characteristics usually differ in affinity basis. For example, various fundamentals of affinity between eluant and analyte include charge or pH, ionic strength, water structure, concentration of specific competitive binding agents, surface tension, dielectric constant, and combinations of two or more of these factors.

a. PH Eluants

Eluants that modify the selectivity of the adsorbent based on pH (i.e. charge) include known pH buffers, acidic solutions, and basic solutions. By washing the analyte attached to this adsorbent with a buffer with a specific pH value, the charge can be modified and, accordingly, the binding force between the adsorbent and analyte in the presence of a buffer with a specific pH value also changes. Those analytes that are less competitive with respect to the adsorbent than others, at a given pH of the eluant, will be desorbed from the adsorbent and elute. In this case, only those analytes that are more strongly bound to the adsorbent at a given pH of the eluant will remain bound.

b. Ionic Strength Eluants

Eluants that modify the selectivity of the adsorbent with respect to ionic strength include solutions of salts of various types and concentrations. The amount of salt solubilized in the eluant solution affects the ionic strength of the eluant and, accordingly, modifies the binding capacity of the adsorbent. Low salt concentration eluants provide a slight modification of the ionic strength of the adsorbent. High salt concentration eluants provide a stronger modification of the ionic strength of the adsorbent.

in. Water based eluants

Eluants that modify the adsorbent selectivity by changing the structure of water or concentrations include urea and solutions of chaotropic salts. Urea solutions are usually used in the concentration range from 0.1 to 8 M. In the preparation of eluants, chaotropic salts, including sodium thiocyanate, can be used. Eluants based on the structure of water modify the ability of the adsorbent to bind analyte due to changes in hydration or structure of bound water. Eluants of this type include glycerin, ethylene glycol and organic solvents. Chaotropic anions increase the solubility of non-polar substances in water, thereby weakening the hydrophobic interactions between the analyte and the adsorbent.

Detergent based eluants

Eluants that modify the selectivity of the adsorbent with respect to surface tension and analyte structure include detergents and surfactants. Acceptable detergents as eluants include ionic and nonionic detergents such as CHAPS, Tween and NP40. Detergent-based eluants modify the ability of the adsorbent to bind analyte based on modifications of hydrophobic interactions when hydrophobic and hydrophilic groups of detergent are introduced. In this case, the hydrophobic interactions between the analyte and the adsorbent, as well as in the analyte, are modified and charged groups are introduced, for example, protein denaturation with ionic detergents such as SDS occurs.

D. Hydrophobic Eluants

Eluants that modify the selectivity of the adsorbent with respect to the dielectric constant are eluants that modify the selectivity of the adsorbent with respect to hydrophobic interaction. Examples of suitable eluants that function in this way are urea (0.1-8M), organic solvents such as propanol, acetonitrile, ethylene glycol and glycerin, as well as detergents similar to those listed above. The use of acetonitrile as an eluant is common for reverse phase chromatography. The introduction of ethylene glycol into the eluant is effective in the elution of immunoglobulins after salt-dependent interaction with thiophilic adsorbents.

e. Combinations of eluants

Suitable eluants can be selected from any of the categories listed above, or they can be a combination of two or more of these eluants. Eluants, which are two or more of these eluants, are able to modify the selectivity of the adsorbent to the analyte based on the multiple characteristics of the elution.

3. Variables of two parameters

The ability to obtain different binding characteristics by choosing different adsorbents and the ability to obtain different elution characteristics by washing with different eluants allow two different parameters to be varied, each of which can individually affect the selectivity with which analytes bind to the adsorbent. The fact that these two parameters can vary widely provides a wide range of binding affinities and elution conditions, so that the methods of the present invention can be used to bind and, accordingly, detect analytes of many different types.

The choice of adsorbents and eluants for the analysis of a particular sample will depend on the nature of the sample, a specific analyte or class of analytes that must be characterized, even if the nature of the analytes is unknown. Typically, the advantage is to obtain a system with a wide variety of binding characteristics and a wide variety of elution characteristics, especially if the composition of the sample to be analyzed is unknown. With the preparation of a system with a wide range of selectivity conditions, the probability of the retention of the analyte of interest by one or more adsorbents increases significantly.

A qualified specialist with experience in chemical or biochemical analysis will be able to determine the selectivity conditions necessary to retain a particular analyte, obtaining a system with a wide range of binding and elution characteristics, and determining the binding and elution characteristics that provide the best analyte resolution. Since the present invention claims systems comprising a wide range of selectivity conditions, the optimal binding and elution characteristics for a given analyte can be determined by a qualified person without undue experimentation.

C. Analites

The present invention enables the resolution of analytes based on various biological, chemical or physico-chemical properties of the analyte by taking these properties into account when choosing the appropriate selectivity conditions. Many of the properties of analytes that can be used to select the appropriate selectivity conditions include a hydrophobic index (or the degree of hydrophobicity of analyte residues), an isoelectric point (i.e., the pH at which the analyte has no charge), hydrophobic moment (or degree of amphipathicity analyte, or the degree of asymmetry in the distribution of polar and nonpolar residues), the lateral dipole moment (or the degree of asymmetry in the distribution of charge in the analyte), the molecular structure factor (the presence of variations in the surface contour of the analyte molecule, for example, due to the distribution of massive side chains along the skeleton of the molecule), components of the secondary structure (i.e., helix, parallel and antiparallel planes), disulfide bonds, exposed electron-donor groups (i.e. His), aromaticity (or the degree of pi-pi interaction between aromatic residues in the analyte) and the linear distance between charged atoms.

Here are examples of types of properties that can be used to resolve a given analyte from a sample by selecting appropriate selectivity conditions in accordance with the claimed methods. Other useful properties of analytes, which can serve as the basis for resolving a specific analyte from a sample, are known or can be determined by a qualified specialist and are considered in the present invention.

The claimed method is not limited in relation to the types of samples that can be analyzed. Samples can be in solid, liquid or gaseous state, although liquid samples are usually used. Solid or gaseous samples are preferably solubilized in an acceptable solvent to obtain a liquid sample in accordance with methods known in the art. The sample may be a biological compound, a non-biological organic compound, or an inorganic compound. The methods of the present invention are particularly useful for resolving analytes in a biological sample, in particular in biological fluids and extracts, as well as for resolving analytes in non-biological organic compounds, especially mixtures of small organic and inorganic molecules.

Analytes can be molecules, multimeric molecular complexes, macromolecular structures, cells, subcellular organelles, viruses, fragments of molecules, ions or atoms. An analyte may be the only component of the sample or belong to the class of structurally, chemically, biologically, or functionally related components that have one or more common characteristics (i.e. molecular weight, isoelectric point, ionic charge, hydrophobic / hydrophilic interactions, etc.) .

Specific examples of analytes that can be resolved using the retent chromatography methods of the invention include biological macromolecules such as peptides, proteins, enzymes, polynucleotides, oligonucleotides, nucleic acids, carbohydrates, oligosaccharides, polysaccharides; fragments of the above biological macromolecules, such as nucleic acid fragments, peptide fragments and protein fragments; complexes of the above biological macromolecules, such as nucleic acid complexes, DNA-protein complexes, receptor-ligand complexes, enzyme-substrate complexes, enzyme-inhibitor complexes, peptide complexes, protein complexes, hydrocarbon complexes and polysaccharide complexes; small biological molecules such as amino acids, nucleotides, nucleosides, sugars, steroids, lipids, metal ions, drugs, hormones, amides, amines, carboxylic acids, vitamins and coenzymes, alcohols, aldehydes, ketones, fatty acids, porphyrins, carotenoids, plant growth regulators, phosphate esters and nucleoside diphosphosugar, such small synthetic molecules as pharmaceutically or therapeutically effective agents, monomers, peptide analogues, steroid analogs, inhibitors, mutagens, carcinogens, antimitotic drugs, antibiotics, ionophores, antimetabolites, analogs of amino acids, antimicrobial agents, transport inhibitors, surface active agents (surfactants), mitochondrial and chloroplast function inhibitors, donors, electron carriers and acceptors, synthetic protease substrates, phosphatase substrates, esterase and lipase substrates protein modifying reagents; synthetic polymers, oligomers and copolymers such as polyalkylenes, polyamides, poly (meth) acrylates, polysulfones, polystyrenes, polyesters, polyvinyl esters, polyvinyl esters, polycarbonates, polyvinyl halides, polysiloxanes, POMA, polyethylene glycols or more copolymers .

III. DATA PROCESSING

The detection of analytes adsorbed by the adsorbent under specific elution conditions provides information about the analytes in the sample and their chemical properties. Adsorption partially depends on the binding characteristics of the adsorbent: analytes that bind to the adsorbent have properties that make binding possible. For example, molecules that are cationic at a specific pH will bind to an anionic adsorbent under elution conditions that include that pH. Strongly cationic molecules will elute from the adsorbent only under very severe elution conditions. Molecules with hydrophobic regions will be bound by hydrophobic adsorbents, and molecules with hydrophilic regions will be bound by hydrophilic adsorbents. Again, the strength of the interaction will depend in part on the degree to which hydrophobic or hydrophilic regions are present in the analyte. Thus, the determination of the fact of binding of certain analytes in a sample by an adsorbent under certain elution conditions not only allows analytes in the mixture, separating them from each other and from analytes that do not have the chemical property necessary for binding, but also determines the class of analytes or individual analytes having specific chemical properties. By collecting information about the retention of the analyte on one or more specific adsorbents under certain elution conditions, one can obtain not only a detailed resolution of the analytes in the mixture, but also chemical information about the analytes, which can lead to their identification. This data is called “retention data”.

The data obtained in retention tests is most conveniently analyzed using a programmable digital computer. A computer program typically contains a readable medium in which codes are stored. Certain codes are dedicated to memory and contain information about localization on the substrate matrix, the identity of the adsorbent and the elution conditions used to wash the adsorbent. Using this information, the program can identify a set of matrix properties that determine specific selectivity characteristics. The computer also contains codes that process signal strength data from various molecular weights obtained from specific predetermined locations on the probe. This data may indicate the number of analytes detected and additionally include signal strength and a specific molecular weight for each analyte.

The computer also contains codes for processing data. The present invention provides a variety of data processing methods. In one embodiment, an analyte recognition profile is provided. For example, retention data for a particular analyte identified by molecular weight can be sorted according to specific binding characteristics, for example, binding by anionic adsorbents or hydrophobic adsorbents. The totality of these data is a profile of the chemical properties of a particular analyte. The retention characteristics reflect the analyte function, which in turn reflects the structure. For example, retention by coordinate covalent metal chelators may reflect the presence of histidine residues in the analyte polypeptide. By combining data on the level of retention on a variety of cationic and anionic adsorbents during elution with varying pH, we can obtain information from which to determine the isoelectric point of the protein. In turn, this parameter reflects the possible number of ionic amino acids in the protein. Accordingly, the computer may contain codes that translate the binding information into structural information. In addition, the secondary processing of the analyte (i.e., post-translational modifications) leads to a change in the recognition profile, which is reflected in differences in binding or mass.

In another embodiment, retention tests are performed with the same set of selectivity thresholds for cells of two different types and retention data of the two tests are compared. Differences in retention charts (i.e., the presence and strength of the signal at any predetermined location) indicate analytes that are differentially expressed in two different types of cells. This may include, for example, the creation of differential maps indicating differences in signal strength in the two retention tests, which indicates that the analytes are more or less retained by the adsorbent in the two tests.

A computer program may also include code that receives instructions from a programmer at the input. A consistent and logical way of selective desorption of analytes from specific predefined locations on the matrix can be foreseen and programmed in advance.

The computer can convert the data to another format for presentation (demonstration). Data analysis may include determination steps, i.e. establishing signal strength as a function of predetermined localization based on the collected data, removing “outliers” (data deviating from the predetermined statistical distribution), and calculating the relative affinity of analyte binding based on retention data.

The data obtained can be presented in various formats. In one format, signal strength is displayed on a graph as a function of molecular weight. In another format, referred to as the "gel format", the signal strength is laid off along the horizontal axis, reflecting the intensity of the darkening, which resembles the appearance of similar messengers in the gel. In another format, signals reaching a certain threshold are represented as vertical lines or stripes on the horizontal axis along which the molecular weight is deposited. Accordingly, each line represents a detected analyte. Data can also be presented in the form of graphs of signal strength for specific analytes, grouped in accordance with the characteristics of the binding and / or characteristics of the elution.

IV. APPLICATIONS OF RETENTANT CHROMATOGRAPHY

Retentate chromatography involves combinatorial separation methods, including the parallel detection and characterization of many analytes. These combinatorial methods have many uses. These include, without limitation, the development of target analyte detection schemes; development of a protein purification strategy; protein purification methods; identification of a specific phage from a phage library that binds a target analyte, including identification of a target epitope using complementary phage libraries; protein purification based on the physicochemical properties of the analyte; monitoring gene expression and differential representation of proteins; screening of substances in toxicology; simultaneous detection of many diagnostic markers; discovery of new drugs; monitoring the assembly of multimeric proteins and detecting in vitro nucleotide translation.

A. Methods for the sequential extraction of analytes from a sample

Retentate chromatography involves analyte retention analysis under a variety of asorbent / eluant conditions. One option for this method is sequential extraction. In sequential extraction, the sample is not treated independently under two different selectivity conditions. Rather, the sample is treated under the first selectivity condition for extracting individual analytes from the sample with an adsorbent, while the non-adsorbed analytes remain in the eluant. Then the sample is processed in the second condition of selectivity. In this case, the extraction of various analytes from the eluant occurs. Often, if the adsorbents used in the first and second cycles have different affinity bases (e.g., normal phase and hydrophobic), then the adsorbents will extract a different set of analytes from the eluant. This second eluant is then treated in the third condition of selectivity, and so on. In the sequential extraction method, the adsorbent is placed on the bottom of the cell, so that the sample can be applied on top of it. The eluant is added to the adsorbent and after binding between analytes in the sample, the eluant is collected. The collected flush is then applied to a second adsorbent and the analytes are extracted from the sample by binding.

According to one embodiment of the invention, the goal of sequential extraction is more preparative than analytical. More specifically, the goal may be to extract from the sample all analytes except those needed. In this case, the sample is very small, i.e. a few microliters on a spot a few millimeters in diameter. Adsorbents are selected so that they do not adsorb analyte, which should not be excluded from the sample. After several cycles, the final collected flush is devoid of unwanted analytes and contains only the analytes needed for subsequent analysis, for example, desorption spectrometry or traditional chromatographic methods.

In another embodiment, an unstoppable sample is analyzed for the presence of analytes by any analytical method. Even after one retention step, this process makes it possible to determine which materials are adsorbed by the adsorbent and which analytes are not.

B. Methods for sequential resolution of analytes in a sample

The goal of retentate chromatography is to unambiguously resolve the amalite from a complex sample mixture. This is especially important for use in clinical diagnostics, the development of new drugs and in functional genomics. In these areas, identification of one or more analytes from a biological sample is required. The present invention provides a method for identifying selectivity conditions under which an analyte is held, and in a repeating, cyclic process, introducing additional binding characteristics or elution characteristics into a selectivity condition that provides improved analyte resolution.

The mass spectrum of a complex sample processed under the condition of selectivity usually contains signals from many components of the sample. The complexity of the signals may interfere with the unambiguous resolution of the analyte. The analyte sequential resolution methods make it possible to identify selectivity conditions with improved analyte resolution for unambiguous detection of the analyte in the sample. The selectivity condition provides an “improved resolution” of the analyte compared to another selectivity condition if the signal from the analyte is more easily distinguishable from the signals of other components. This includes, for example, reducing the number of analytes associated with the adsorbent, thereby reducing the total number of signals, or increasing the selectivity of the selectivity condition for the analyte, amplifying the analyte signal in comparison with other signals. Of course, when the analyte binds exclusively to the substrate, only the analyte signal is generated during detection.

Sequential resolution methods involve a repeating process in which additional selectivity characteristics (binding or elution) are sequentially introduced into a constant set of selectivity characteristics at which the analyte is retained. At the first stage, a number of selectivity conditions are checked to identify one in which the analyte target is held. In the next step, one or more selectivity characteristics are selected for a constant set of selectivity conditions used for further analysis.

So get a new set of conditions of selectivity. Each of the conditions of the new set includes selected characteristics from the constant set and at least one new condition that is not included in the constant set. For example, if the constant kit includes an anionic adsorbent and a low salt eluant, then a new condition may involve varying the pH of the eluant. Each of these new variables is tested for its ability to improve analyte resolution and one modified selectivity condition is determined that provides improved resolution. In the next step, this additional condition of selectivity is introduced into a constant set.

The modified constant set is again checked in a similar manner, obtaining a set of new selectivity conditions that includes characteristics from the constant set and new characteristics. Thus, at each stage, the selectivity conditions are chosen so that the resolution of the analyte is improved compared with the selectivity condition used in the previous step.

The method is well illustrated by an example. A cell sample usually contains hundreds or perhaps thousands of proteins. Assume that the task is to unequivocally resolve a single target analyte, a protein from a given sample. In the first step, a retention card for the target analyte is obtained using a variety of selectivity conditions. For example, the adsorbents may be an anion exchanger, a cation exchanger, a normal phase adsorbent, and a reverse phase adsorbent. The elution conditions tested on each adsorbent can vary with respect to pH, ionic strength, concentrations of certain detergents and degree of hydrophobicity. For example, four different elution conditions can be tested for each selectivity condition. Thus, in this case, sixteen different selectivity conditions are tested for the ability to adsorb the analyte target.

Based on the retention map, at least one selectivity condition can be selected under which the analyte target is held. You can select the selectivity condition under which the target binds as much as possible. However, the choice of a selectivity condition under which the target binds non-maximally can be advantageous if this selectivity condition is more selective with respect to the target than other selectivity conditions. Suppose, for example, that the analysis of the retention map shows that the target is held by anion-exchange adsorbents at pH close to neutral, but also weakly adsorbed by a hydrophobic adsorbent.

One of the variable adsorbents or eluants from the selectivity condition determined as a result of analyte retention is selected for use in all subsequent selectivity conditions. Thus, it is added to the “set of constants of selectivity conditions”.

In the next cycle, the ability of the target analyte to bind under the second set of selectivity conditions is tested. Each selectivity condition in the second set includes elements of the selectivity conditions from the constant set. However, each selectivity condition also includes a different variable - another adsorbent or eluant, introduced selectivity condition. Thus, taking into account the need to use at least one set of constants, the second set of selectivity conditions is also chosen so that it is more diverse than the first set. Methods of increasing diversity include, for example, checking finer gradations of the elution condition or changing the strength of the adsorbent. They may also include, for example, the introduction of selectivity characteristics into the conditions of selectivity.

Continuing with the example, an anion exchange adsorbent can be added to the set of constants. Now this condition is checked with a wide variety of variables, i.e. eluants or adsorbents. The tested eluants may include various buffers with low pH values with finer gradations than those checked in the first cycle. For example, in the first cycle, you can check the buffers with pH 3.0, pH 5.0, pH 7.0 and pH 9.0, and see that the target is that the target is held by an anion exchange adsorbent at pH values close to neutral. During the second cycle, the tested buffers can have pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5 and pH 8.0. In addition, each of these buffers may vary in other elution characteristics, i.e., ionic strength, hydrophobicity, etc.

Analysis of the second retention card obtained at this stage usually allows one to determine a condition that provides improved analyte resolution than the selectivity condition identified at the first stage. Again, one of the variables of this selectivity condition is selected and the selectivity conditions are introduced into the set of constants for further testing during the next cycle.

Continuing the example, suppose that the selectivity condition in the second cycle in which the analyte is resolved includes a buffer with a pH of 6.5. This eluant can now be introduced into a set of constants, which now includes an anion exchange resin and a buffer with a pH of 6.5. During the next cycle, this selectivity condition is introduced into a constant set, and the following selectivity condition is varied. Variables may include, for example, the introduction of a new component in an eluant that changes ionic strength; or another adsorbent can be introduced into the mixture, so that various hydrophobic adsorbents are mixed with the anion-exchange adsorbent; or it is possible to vary the density of the anion exchange resin. Again, a selectivity condition that provides improved analyte resolution is identified from this set.

The process can continue until the analyte is purified to the required homogeneity. In this case, the selectivity condition is specific for the analyte.

As can be seen, by increasing the number of variables used at each step, the number of cycles necessary to identify an acceptable selectivity condition can be reduced.

B. Methods for preparative purification of the analyte

As one aspect of the present invention, methods for purifying an analyte are claimed. These methods take advantage of retentate chromatography to quickly identify the basis of affinity for analyte adsorption. At the first stage, the analyte is processed under a variety of selectivity conditions to determine analyte retention under these conditions by retentate chromatography. As a result, an analyte recognition profile is obtained. The selectivity conditions under which the analyte is retained is used to develop a protocol for preparative purification of the analyte.

For preparative purification of the analyte, the analyte is sequentially adsorbed and eluted on a series of adsorbent / eluant combinations for which analyte binding is indicated. So, for example, a recognition card may indicate that the analyte is bound by a normal phase adsorbent and a metal chelator. The analyte is then applied to a first chromatographic column containing, for example, a normal phase adsorbent that binds the analyte. Unbound material is washed. The analyte is then eluted with a sufficiently rigid wash. The eluant is applied, for example, to a column with a metal chelator, which binds the analyte. Unbound material is washed. The bound analyte containing material is then eluted from the metal chelating column. Thus, the analyte is isolated in preparative amounts. The preparative amount of the sample is at least 10 μl, at least 100 μl, at least 1 ml, at least 10 ml.

The information obtained during the sequential resolution of analytes can be used to develop a strategy for large-scale chromatographic (based on elution) protein purification. The basis of adsorbent affinity, binding conditions, and elution conditions (i.e., selectivity conditions) for the target protein analyte are determined by retentate chromatography. Obtaining such information saves a tremendous amount of time, energy and expensive analytes that would otherwise be spent on trial and error in developing a cleaning strategy. This section also describes examples of preparative purification carried out with commercially available adsorbents.

D. Methods of preparation of probes for specific detection of analytes

The present invention claims probes for the specific detection of one or more analytes by desorption spectrometry, as well as methods for producing such probes. Such analyte-resolving probes are useful for specific detection of analytes in the implementation of diagnostic and analytical methods.

The first step in obtaining a probe for resolving one or more analytes is to obtain a retention map of the analytes in many different adsorbent / eluant combinations. For example, analyte resolution can be set for four different adsorbents washed with one of five different eluants. An analysis of the retention card obtained will show which condition or conditions of selectivity are best resolved by analytes. It is preferable to identify one selectivity condition that uniquely resolves all analytes. One or more selectivity conditions are then selected for use in the analyte-resolving probe so that each analyte resolves on at least one spot of the adsorbent. The probe may also contain an adsorbent that does not bind analyte or analytes. This adsorbent spot is used as a control. The probes may contain a plurality of adsorbent spots in predetermined locations selected by their ability to retain and resolve analyte or analytes. In this case, the adsorbents are chosen so that the analyte is bound under the condition of treatment with one eluant. This may be useful, because during the detection process, the entire probe can be washed with one eluant.

The retention card obtained for a particular analyte can be used to create a special, “custom” adsorbent for this analyte. For example, the nature of the analyte-retaining adsorbents indicates a set of analyte affinity basics. A “custom” adsorbent can be constructed by preparing a multiplex adsorbent including adsorbent elements that provide these affinity basis. Such a “custom” adsorbent is very selective for the target analyte. One or only a few “custom” adsorbents can provide an amalite recognition card. For example, if it is shown that under certain conditions of elution, the analyte is retained by adsorbents that bind materials with a certain degree of hydrophobicity, positive charge and aromaticity, then you can create a “custom” adsorbent combining functional groups with combinatorial synthetic methods that will provide all three of the above properties. The analyte is identified by detecting binding to such an adsorbent.

Such probes are useful for detecting analyte or analytes in a sample. The sample is treated under selectivity conditions and the probe is examined by desorption spectrometry. Since the probe allows analytes, their presence can be determined by the characteristics of the recognition profile. Such probes are particularly useful for identifying a set of diagnostic markers in a sample obtained from a patient.

According to one embodiment of the invention, a matrix is constructed for binding proteins of interest of a particular class. These include diagnostic markers, as well as analytes with a specific function. For example, you can get a matrix that will specifically bind cell surface proteins, enzymes of a certain class (e.g. kinases), transcription factors, intracellular receptors, etc. Adsorbents may be specific for biopolymers, for example antibodies.

In one embodiment, the adsorbents are organisms such as phage that carry protein ligands to proteins of a particular class on their surface. In this case, the phage library can be screened with molecules of a certain class in order to exclude phages that bind molecules of this class. Then the phage that has been isolated from the population in this way are used as adsorbent.

D. Diagnostic probes and diagnostic methods

When diagnosing pathological conditions, it is often required to detect one or more molecular markers of the disease in a sample taken from a patient. Many diseases can be diagnosed by the presence of a single diagnostic marker. Diagnosing other diseases may require the detection of multiple diagnostic markers. In addition, the detection of several markers can increase the reliability of the diagnosis. Accordingly, the present invention claims probes for desorption spectrometry containing at least one adsorbent that is capable of resolving at least one diagnostic marker of a pathological condition.

Obtaining such probes involves, first of all, selecting the markers to be detected. Such a marker can be a marker of any disease state, i.e. cancer, heart disease, autoimmune disease, viral infection of Alzheimer's disease or diabetes. For example, detection of a prostate-specific antigen (PSA) is highly likely to indicate prostate cancer. HIV infection can be diagnosed by detecting antibodies to several HIV proteins, such as one of p17, p24 or p55 and one of p31, p51 or p66 and one of gp41 or gp120 / 160. Detection of amyloid-b42 or tau protein in the cerebrospinal fluid is highly likely to indicate Alzheimer's disease. Markers can also be identified using the claimed methods for establishing the differential presence of analyte in healthy subjects as opposed to patients.

In the next step, adsorbents are obtained that hold one or more diagnostic markers. It is preferable to obtain a single adsorbent that would resolve all markers. This can be achieved, for example, by creating a spot containing several antibodies, each of which binds one of the markers of interest. Alternatively, the probe may contain many spots of adsorbent, with each spot under these conditions of selectivity can resolve at least one analyte target. According to one embodiment of the invention, the adsorbent is a multiplex adsorbent containing ligands that are specific for markers. For example, the adsorbent may comprise an antibody, a polypeptide ligand, or a polynucleotide that specifically binds a target analyte. In one embodiment, the antibody is a single chain antibody identified by screening for a combinatorial library. Single chain antibodies specific for specific markers can be obtained by screening phage libraries in accordance with the methods described in this application.

According to another embodiment of the invention, the adsorbent contains inorganic biomolecular components that either specifically retain the target analyte or retain the analyte with a specificity sufficient for unambiguous resolution by desorption spectrometry. The preparation of adsorbents for the detection of specific analytes is also described in this application.

It is important that the single adsorbent spot used in these methods should not be specific to a single analyte, and therefore, no specific affinity between the target and the adsorbent due to the biopolymer is required. Earlier affinity detection methods relied primarily on specific binding between the biopolymer and the target. Such types of binding include, for example, the specific affinity of an antibody for a protein, a polynucleotide for a complementary polynucleotide, or pectin for a carbohydrate. Such specificity was necessary due to indirect detection methods: not a target was identified, but a label, often associated with an adsorbent. Accordingly, the more specific the adsorbent, the less likely that the impurities will bind to the adsorbent and prevent specific detection. However, desorption spectrometry detects the analyte directly. Accordingly, the presence of impurities does not affect the specificity of detection, unless the signal from the impurity does not overlap the signal from the target.

Diagnostic methods include, first of all, obtaining from a patient a sample for testing. A sample, for example, can be tissue, blood, urine, feces or other body fluids (lymph, cerebrospinal fluid, intraarticular fluid, etc.). The sample is then brought into contact with a substrate containing diagnostic adsorbents under conditions that make it possible to retain diagnostic markers. The adsorbent is washed with an appropriate eluant. Markers are then detected (i.e., resolved) by desorption spectrometry (i.e., mass spectrometry).

The present invention also claims kits for specific detection of diagnostic markers, comprising (1) a substrate for desorption spectrometry containing at least one adsorbent in at least one predetermined location that resolves at least one diagnostic marker in a selectivity condition that includes an adsorbent and an eluant, and (2) eluant or instructions for preparing the eluant. When bringing the sample into contact with the adsorbent and washing with eluant, i.e. when the selectivity condition is fulfilled, the analyte is purified sufficiently or specifically binds, which makes it possible to resolve it by desorption spectrometry.

E. Methods for the identification of proteins

As another aspect, the present invention provides a method for identifying proteins. This method provides for the determination of matching parameters in the physicochemical characteristics of the protein analyte using retentate chromatography and screening of protein databases to identify proteins with matching parameters. The processing of physico-chemical information based on retention characteristics was discussed above. An amino acid sequence and / or a nucleotide sequence encoding the amino acid sequence of each protein can usually be extracted from databases. It is easy to derive structural characteristics from this information, such as molecular weight, hydrophobicity, pI, fragment mass, etc. The analyte protein will have a specific structural characteristic that is common only with some subset of proteins available in the database. Accordingly, the identification of possible candidate proteins is carried out on the basis of protein sorting by those structural characteristics that are common with the analyte protein. Thus, taking into account inaccuracies, the degree of specificity, or the level of confidence inherent in the establishment of one or more physicochemical properties, one cannot expect that the database proteins will exactly match the standard in all respects. Accordingly, matching parameters can be used to establish, for example, the proximity of the characteristics of the protein analyte and the characteristics of the reference polypeptides in the database.

With an increase in the number of identified genome genes, the likelihood that any protein analyte is represented in the databases as a reference polypeptide increases.

Accordingly, the claimed method makes it possible to quickly resolve the protein of interest in the sample to obtain structural information about the protein and then use this information to identify the protein.

G. Methods of assembly of multimeric molecules

The ability of adsorbents to retain the desired molecules can be used in the construction of multimeric molecules and to obtain compounds that affect such an assembly. A unit of a multimeric molecule is attached to an adsorbent. Then it is brought into contact with a sample containing another unit of a multimeric molecule. Such contact can be carried out in a variety of conditions, which allows you to control the binding conditions. The attachment of the subunit to the multimer can be controlled by desorption spectrometry. Then in the same way you can control the accession of subsequent subunits. The methods for screening potential drugs described herein are useful in testing agents for their ability to interfere with the assembly of multimeric molecules. Accordingly, the analyte at one stage of the process becomes an adsorbent at another stage.

3. Methods of setting enzymatic tests

The present invention also provides methods for staging enzymatic tests. Enzymatic tests usually involve contacting a test sample with an enzyme substrate under conditions in which the enzyme is active. After exposure of the enzyme to the substrate, the amount of the ezimatic reaction product is evaluated. Quantitative analysis determines the amount of product. This amount is usually compared with a control or standard curve, thereby determining the level of enzymatic activity in the sample.

The present invention provides a method for detecting an enzyme, including determining the level of enzymatic activity in a sample. The advantage of the method is the fact that the activity of the enzyme often leads to the formation of a product whose mass is different from the mass of the starting substrate. When implementing the method, a solid phase is prepared containing an adsorbent that binds the substrate. The amount of substrate bound to the adsorbent is fixed. Then the adsorbent is brought into contact with the sample for such a time and under conditions that allow the enzyme to act on the substrate.

Any bound material is detected by desorption spectrometry. Detection of an analyte with a molecular weight characteristic of the product of enzymatic activity indicates the presence of the enzyme. The signal strength will be a function of the level of enzymatic activity in the sample.

I. Methods for the identification of analytes that are expressed differentially in various biological materials

As one aspect of the present invention, methods for identifying organic biomolecules, especially proteins that are differentially expressed in two or more samples, are claimed. "Differential expression" means differences in the quantity or quality of an analyte between two samples. Such differences can occur at any stage of protein expression, starting with transcription and ending with post-translational modification. These methods have the advantages of extremely high resolution retentate chromatography. First, for analytes contained in two samples, recognition profiles are obtained using the same set of selectivity conditions. The larger the number of selectivity conditions used, the higher the resolution of analytes in the sample and, accordingly, a larger number of analytes can be compared. Recognition cards are then compared to identify analytes that are differentially held by two sets of adsorbents. Differential retention (retention) implies quantitative retention. This indicates increased or decreased expression. Differential retention also indicates qualitative differences in the analyte. For example, differences in post-translational modification of a protein can lead to differences in recognition maps, detected as differences in binding characteristics (for example, if a protein is glycosylated, it will otherwise bind to lectin adsorbents), or as differences in masses (for example, as a result of differences in post-translational cutting). The analysis can be performed on a programmable digital computer.

The method is particularly useful for detecting genes that are differentially expressed in two different types of cells. Such pairs of cells can be represented by normal and pathological cells, for example, cancer cells, or cells that are at different stages of development or differentiation, or at different stages of the cell cycle. However, the method is also applicable for the study of two cells of the same type under different conditions. For example, the method is applicable in toxicology for screening and testing agents for their ability to modulate gene expression in a cell. In this case, one biological sample is brought into contact with the test agent, and the other is not. Then retentate maps of the samples are compared. The method may indicate a fact of reduced or increased expression of a protein or other biomolecule, or of changes resulting from differing retention or mass changes.

Using information on the physicochemical properties of differentially expressed proteins obtained from retention charts, using the methods described, candidate proteins for identity can be determined.

This method is applicable for the identification of diagnostic markers of diseases. Proteins that are differentially expressed in samples taken from patients or in cultured cells of patients compared to normal samples or cells may be diagnostic markers. In general, it is preferable to compare normal samples taken from a statistically significant sample of patients. Thus, information can be collected to identify diagnostic markers common to all or many individuals suffering from a given pathology.

K. Sensitivity enhancement by amplification of a catabodic signal

The sensitivity of detecting the differential presence (resulting from differential expression) in a complex mixture can be significantly increased by fragmenting a large protein into small pieces and detecting precisely these fragments. Sensitivity increases due to several factors. First, when all the proteins in a sample are fragmented, for example, by enzymatic digestion, large proteins form more fragments than small proteins. Secondly, the overall sensitivity of desorption spectrometry is higher than the lower molecular weights of the analytes. Thirdly, protein fragmentation increases the number of signals from the target, thereby increasing the probability of target detection. Fourth, protein fragmentation increases the likelihood of retention and, therefore, the detection of at least a protein fragment. Fifthly, if protein is represented differently in two samples, then an increase in the number of signals increases the likelihood of detecting differences in the quantity.

Usually they seek to reduce the complexity of the analyte mixture before analysis. Fragmentation increases complexity.

Accordingly, according to one embodiment of the invention, the detection sensitivity of the analyte is increased by converting the analyte into fragments of lower molecular weight before detection. Fragmentation can be accomplished by any methods known in the art. For example, protein analytes can be fragmented by endoproteases. Nucleic acids can be fragmented using endonucleases. The sample can be fragmented before or after application to the adsorbent.

L. Methods for the identification of ligands for the receptor

In functional bonds in biological systems, interactions between the receptor and the ligand are often used. For example, when transcription is activated, the ligand is preliminarily pre-linked to the transcription factor. In many pathological conditions, an abnormal interaction takes place between the receptor and the ligand. Impaired receptor and ligand binding is often the goal when developing new drugs. However, the ligand to a specific receptor is often unknown; this receptor is called "orphan".

The present invention provides a method for applying retentate chromatography to identify ligands for receptors. The method involves attaching a receptor to an adsorbent. Then, a sample in which the presence of the ligand to the receptor is assumed is brought into contact with the attached receptor under an elution condition that promotes binding between the receptor and the ligand. Ligands bound to the receptor are then detected by desorption spectrometry. The resolution of this method in part is due to the sensitivity of desorption spectrometry when detecting small amounts of material attached to the adsorbent.

Attachment of the receptor to the adsorbent requires the identification of an adsorbent that retains and, preferably, specifically binds the receptor. The present description provides methods for identifying adsorbents that specifically bind a protein. According to one of the methods, the adsorbent contains an antibody specific for the receptor. In another embodiment, the receptor is prepared as a recombinant fusion protein containing an entity for specific binding. For example, a receptor may be fused to an Fc fragment of an antibody. This fragment binds protein A, which can be integrated into the adsorbent.

The choice of sample for ligand detection is determined by the experimenter. For example, if the receptor is a nuclear receptor, then the sample may be a nuclear extract. If the receptor is a cytoplasmic receptor, then the sample may be a liquid washing the surface of the cells, for example, serum in the case of a receptor on the surface of epithelial cells.

Typically, a sample is incubated with the receptor under physiological conditions for the time necessary for binding, for example, several hours at 37 ° C. Then unbound material is washed. This method enables the rapid identification of ligands, which requires months using conventional methods.

Retentate chromatography allows parallel processing of samples on several spots of adsorbent. Accordingly, the method may include testing many different samples for the presence of a ligand, as well as testing a single sample in a variety of incubation and elution conditions.

By determining the mass of the identified ligand and the various physicochemical properties of the ligand, ligands can be identified using information from genomic databases.

According to another embodiment of the invention, a set of probes is prepared on which proteins from the cell are retained. Such probes are useful as secondary probes to identify molecules from cells that

bind to retained molecules. After obtaining a retentate card based on the probe, the probe is re-contacted with the test material, usually under less stringent conditions than those used to obtain the secondary probe, and predefined locations are analyzed. The molecules that bind to previously retained molecules turn out to be newly bound.

M. Methods for identifying new drugs

The identification of molecules that prevent binding between the receptor and the ligand to it is an important step in the development of new drugs. The present invention provides methods for screening compounds for their ability to modulate binding between an adsorbent and an analyte (i.e., an adsorbent receptor and an analyte ligand) by contacting the adsorbent and analyte with a test compound and detecting binding between the adsorbent and analyte by desorption spectrometry.

Rapid screening of combinatorial libraries for potential drugs requires the ability to test the interactions of thousands of drugs and the identification of agents that inhibit this interaction or, conversely, facilitate it. Retentate chromatography makes it possible to attach one member of a ligand / receptor pair to a substrate and use it as a secondary adsorbent. Then, after bringing the couple member into contact with his partner and agent, desorption spectrometry can determine if there is a connection between the partners and how strong it is. The advantages of retentate chromatography in screening methods include the possibility of specific binding of the receptor to the substrate by means of an adsorbent, the ability to quickly place the receptor on many spots of adsorbent for parallel processing, and the processing speed, which is achievable when taking into account the results of desorption spectrometry.

1. Screening test

The method involves obtaining an adsorbent; bringing the adsorbent into contact with the target analyte in the presence or absence of an agent in one or more conditions of selectivity and determining the level of binding in the presence and absence of an agent. The level of binding is determined by retentate chromatography (i.e., obtaining a recognition profile). The experiment can be carried out with control without adding an agent or with control when another quantity or type of agent is added, and a zero amount is determined by extrapolation. A statistically significant difference in the level of binding (p <0.05) indicates that the agent modulates binding.

This method is especially useful for screening analytes (i.e. proteins) - drug candidates. After obtaining a retention map of proteins or a recognition profile from serum or any other target cells, the agent is contacted with a matrix on which analyte is held in predetermined locations. After binding has occurred, the unbound agent is eluted or washed. Those analytes that hold the bound agent under selectivity conditions are identified directly by desorption spectrometry, since the agent itself is a new component of the retention card (i.e., the agent is desorbed and detected directly). This method is especially suitable for screening potential drugs, both agonists and antagonists, by their ability to bind analytes or simulate one or more biological processes.

2. Receptor and ligand

The adsorbent and the target analyte need not be involved in specific binding. However, in particularly useful methods, the adsorbent and target analyte are a ligand / receptor pair.

In one embodiment, the members of the ligand / receptor pair are a hormone and a cell surface receptor or intracellular receptor. The adsorbent may be a whole cell or cell membrane in the case of a receptor associated with the membrane. A protein receptor or other potential target drug can be used as an adsorbent for screening combinatorial drug libraries. Hundreds or thousands of potential drugs can be applied to one type of receptor and predetermined localization on the adsorbent. After removal of unbound or weakly bound potential drugs (i.e., agents), bound agents are detected and identified by desorption spectrometry.

According to another embodiment of the invention, the adsorbent is an enzyme that binds to and modifies the target substrate. Agents screen for the ability to modulate the enzymatic transformation of the analyte. For example, enzymatic activity can be detected because the analyte recognition profile may differ from that for the product of enzymatic activity. Differential retention indicates a change in agent binding.

The receptor / ligand can be retained on the substrate in various ways. According to one method, the receptor / ligand is directly retained by a non-specific adsorbent. According to another method, the adsorbent is specific for the receptor / ligand. For example, the adsorbent may contain an antibody specific for the receptor / ligand. The receptor / ligand may be a fusion protein in which one of the fragments specifically binds an adsorbent, for example, as an Fc fragment binds protein A. According to one of the methods, an organism is attached to a substrate, for example, a phage from a phage library that has a polypeptide specifically binding on its surface receptor / ligand. The ligand is captured by the polypeptide. The adsorbent may also be an analyte already retained on the substrate, i.e. may be a secondary adsorbent, a quaternary adsorbent, etc.

The present invention claims a particularly useful method for assessing both the direct and indirect effects of binding a drug (or other agent) to a target. The detection of one or several analytes on a retentate map obtained for the proteins of certain target cells can be changed by the action of an agent (i.e., a potential drug) to 1) the target protein itself, 2) some other analyte ( non-binding drug protein), or 3) for gene expression (or its suppression). It is the high resolution and high information content of retentate chromatography that makes it possible to detect such changes, i.e. differences between retentate maps or recognition profiles in the presence and absence of the drug induced by the drug, making this method one of the most powerful tools available for proteomics, functional genomics, development of new drugs, monitoring of therapeutic drugs and clinical diagnostics.

3. Testing agents

A test agent, which should be tested for its ability to modulate the expression of protimosin, is administered to an experimental animal or to cells cultured in vitro. The choice of test agent is the responsibility of the experimenter. However, due to their diversity and ease of administration as pharmaceuticals, small molecules are preferable as test agents.

a. Chemistry

Testing agents can be obtained from various sources. For example, combinatorial libraries of molecules are available for screening. Using such libraries, thousands of molecules can be screened for regulatory activity. According to one preferred embodiment of the invention, highly effective screening methods provide a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened using one or more methods similar to those described here, in order to identify those representatives of the library (specific chemical species or subclasses) that have the desired characteristic activity. The compounds thus identified serve as conventional “lead compounds” or can themselves be used as potential or true drugs.

The preparation and screening of combinatorial chemical libraries is well known in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (See, e.g., U.S. Patent 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37: 487-493, Houghton et al., (1991) Nature, 354: 84-88). Peptide synthesis is certainly one of the approaches applicable to the practice of the present invention. Other chemical approaches to obtain libraries of chemical diversity may also be used. Such chemical compounds include, but are not limited to: peptoids (PCT Publication No. WO 91/19735, December 26, 1991), encoded peptides (PCT Publication WO 93/20242, October 14, 1993), random bio-oligomers (PCT Publication WO 92 / 00091, January 9, 1992), benzodiazepines (US Pat. No. 5,288,514), sabotomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc. Natl. Acad. Sci. USA 90: 6909-6913) , vinyl polypeptides (Hagihara et al., (1992) J.Amer.Chem.Soc. 114: 6568), non-peptide beta-glucose-coated peptidomimetics (Hirschmann et al., (1992) J.Amer.Chem.Soc . 114: 9217-9218), analogues of organic synthe from libraries of small compounds (Chen et al., (1994) J. Amer. Chem. Soc. 116: 2661), oligocarbamates (Cho et al., (1993) Science 261: 1303), and / or peptidyl phosphanates (Campbell et al., (1994) J. Org. Chem. 59: 658). See, Gordon et al., (1994) J. Med. Chem. 37-.1385, nucleic acid libraries, peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g. , Vaughin et al., (1996) Nature Biotechnology, 14 (3): 309-3144 and PCT / US96 / 10287), carbohydrate libraries (see, e.g., Liang et al. (1996) Science, 274, 1520- 1522 and U.S. Pat. and 5,519,134, morpholine compounds US Patent 5,506,337, benzodiazepine 5,288,514 and the like).

Combinatorial library retailers are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech. Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford , MA).

H. Methods of preparing agents that specifically bind analyte

The present invention provides methods for producing agents, for example, single chain antibodies that specifically bind a target analyte.

Such agents are useful, for example, as specific diagnostic agents for attachment of targets in the study of ligand / receptor interactions. This method is especially useful for producing agents against targets that can be obtained in such small quantities that it is practically impossible to obtain antibodies by immunization of animals. The method involves the steps of obtaining a substrate with a target attached to it; obtaining a library of organisms carrying screening agents; bringing the library into contact with the target for specific retention of organisms by interacting with the target and detecting retained organisms by desorption spectrometry.

These steps can be carried out in parallel with a large number of adsorbent analyte candidates in large populations without loss due to transfer and uncertainties associated with separate selection and detection procedures, including amplification and labeling for indirect detection.

1. Preparation of substrate

At the first stage, the method provides for the preparation of a substrate containing an adsorbent that will serve as a target for the polypeptide agent presented in the library to be screened. In one embodiment, a substrate is prepared with a target adsorbent already attached. According to another embodiment of the invention, a substrate is prepared with an adsorbent that binds the analyte target, the adsorbent is brought into contact with the analyte under conditions of elution that ensure analyte retention, and the adsorbent target is used as a target for the library. According to one embodiment of the invention, the target is differentially expressed in two types of cells to be compared. For example, targets may be derived from differentially expressed mRNAs or may be differentially expressed proteins. Methods for identifying such differentially expressed proteins by retentate chromatography methods are described above.

Once a differentially expressed analyte protein is identified, a selectivity condition can be determined in which the analyte is uniquely resolved. More preferably, analyte retention should be specific or exclusive. The analyte sequential resolution methods described above make it possible to identify the selectivity conditions under which specific binding of the target analyte from a complex sample occurs. According to one embodiment of the invention, the bound target may be modified, for example, by treatment with an enzyme.

Alternatively, the method may begin at the mRNA or EST stage. In this case, differentially expressed mRNAs or ESTs are identified by routine methods. These molecules are then transcribed and translated in vitro and in situ for binding to the adsorbent. For example, a substrate is prepared for desorption spectrometry with many spots of adsorbent. The substrate is applied to a cylindrical tube, thereby obtaining a cell at the bottom of which is an adsorbent. Reagents for transcription and translation in vitro of differentially expressed mRNAs (usually in the form of cDNA) are placed in the cell. (For methods, see, e.g., Sambrook et al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) and Current Protocols in Molecular Biology, FM Ausubel et al., Eds. , (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.)). Translation of mRNA or EST leads to the formation of a polypeptide that is adsorbed. The cylindrical tube is removed, and the spots of the adsorbent are washed with the eluant, identifying the selectivity condition under which the analyte polypeptide is retained.

2. Getting libraries

The second stage involves obtaining libraries. Libraries contain organisms (genotypes) that carry any combinatorial peptide libraries (“polypeptide agents”) on their surfaces. However, single-stranded antibodies are attractive because they can subsequently be used in immunoassays.

Many libraries and methods for using them are known in the art. The basic concept of such methods is to establish a physical association between the polypeptide ligand to be screened and the polynucleotide encoding the polypeptide. This physical association is ensured by the multimeric molecular complex, in this case, by the body, i.e. a phage particle that carries a polypeptide as part of a capsid containing the phage genome in which the polypeptide is encoded. The establishment of a physical association between polypeptides and the genetic matrix encoding them enables the simultaneous mass screening of a very large number of organisms carrying different polypeptides. Organisms carrying a polypeptide that has affinity for the target bind the target, and these organisms are enriched in affinity screening of the target. The identity of the polypeptides present on these organisms can be determined based on their genomes. Using these methods, a polypeptide for which target affinity is shown can then be synthesized in large amounts by conventional methods.

The organisms that are most often used to obtain libraries include bacteriophage, in particular filamentous phage, and, in particular, phages M13, Fd and F1. In most works, the genes gIII or gVIII of these phages were used to represent polypeptides in libraries with the formation of fusion proteins. See, Dower, WO 91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gene III); Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII). See also Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382 (1990); Devlin et al., Science 249, 404-406 (1990), Scott & Smith, Science 249, 386-388 91990); Ladner et al., US 5,223,409 and Ladner et al., US 5,571,698. Such fusion proteins contain a signal sequence typically belonging to a secreted protein other than a surface phage protein, as well as a gene III or VIII protein or fragments thereof. Exogenous coding sequences are often inserted near the N-terminus of gene III or VIII, although other insertion sites are possible. Separate filamentous phage vectors were constructed to produce a second copy of gene III or VIII. In such vectors, exogenous sequences are represented by only one or two copies. Expression of the second copy effectively reduces the proportion of fusion protein incorporated into phage particles, and can be an advantage in reducing selection for polypeptides that are unfavorable for phage growth. The presence of antibody fragments on the surface of viruses infecting bacteria (bacteriophage or phage) makes it possible to obtain human sFVs with a wide range of affinities and kinetic characteristics.

In another embodiment, exogenous polypeptide sequences are cloned into phagemid vectors encoding a phage surface protein and phage packaging sequences, but are unable to replicate. Phagemids are transfected into cells and packaged by infection with helper phage. The use of the phagemid system also has an effect on the dilution of fusion proteins formed from a surface protein and the polypeptide represented by copies of a wild-type surface protein expressed by a helper phage. See nanp., Garrard, WO 92/09690.

Eukaryotic viruses can be used to represent polypeptides in a similar way. For example, the presentation of human heregulin fused to gp70 of Moloni mouse leukemia virus has been described by Han et al., Proc. Natl. Acad. Sci. USA 92: 9747-9751 (1995). Spores can also be used as replicating organisms. In this case, the polypeptides are presented on the outer surface of the spores. For example, B.subtilis spores have been shown to be acceptable. The surface protein sequences of such spores are given by Donovan et al., J. Mol. Biol 196, 1-10 (1987).

Cells can also be used as replicating organisms. Representative polypeptides are inserted into a gene encoding a cellular protein that is expressed on a cell surface. Preferred bacterial cells include Salmonella typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Bacteroides nodosus, Maraxella bovis and, in particular, Escherichia coli. The outer surface proteins are discussed in detail by Ladner et al., US 5,571,698 and Georgiou et al., Nature Biotechnology, 15,29-34 (1997). For example, the E. coli lamB protein is convenient.

3. Library screening

The third step involves screening the library to identify the ligand that specifically binds the target. The substrate carrying the target is brought into contact with a library of polypeptide agents under elution conditions that correspond to the specific binding between the polypeptide and the target molecule. Organisms presenting agents that recognize the target bind the target already attached to the substrate. Removal of unbound particles and retention of bound particles is achieved by certain elution conditions.

A body population, in this case an M13 phage containing approximately 10 ¹¹ plaque forming units (combat) per ml, is applied onto a matrix substrate with predetermined localizations containing bound target adsorbents (i.e. proteins). Upon contact, a selectivity condition is selected that is optimized for the target adsorbent (i.e., selectivity threshold modifier or eluant) so that only a small fraction of all phage genotypes are selectively retained, preferably less than 5-10. Unbound phage (i.e., a phage that did not bind to the target adsorbent) and a phage weakly bound to the target adsorbent are washed with an eluant that disrupts all analyte / adsorbent-target interactions, except for the most selective ones. Phages that carry polypeptides having the highest affinity for the target adsorbent are selectively retained.

4. Detection of retained organisms containing agents that specifically bind the target

At the fourth stage, the binding of organisms to the target is detected by desorption spectrometry. For example, phage M13 has thousands of copies of a single surface protein. When phage is treated with laser radiation during desorption spectrometry, surface proteins detach and become detectable. Thus, it can be determined whether the phage library contains a carrier agent that binds to the target. To obtain organisms for further research, the screening step can be carried out in parallel at different locations on the probe, or the substrate can be of such physical dimensions that are large enough so that the laser does not disconnect all organisms bound to the surface. This method is especially sensitive because it can detect even several phage particles associated with the analyte.

In the case of phage M13, the preferred detection method involves controlling by desorption spectrometry the appearance of the surface protein of gene VIII as a signal of the marker protein. Thus, we found “positive” target adsorbents with which only 5 phage particles (battle) were associated (the number of phage particles was calculated from known dilutions). Other phage markers, in order of preference, include gene V, gene X, and gene III (including fusion products thereof).

After determining the predetermined localizations with the most affinity adsorbents, i.e., those localizations on the matrix, on which, after processing under conditions of high selectivity (i.e., strong eluants), the smallest number of phage particles is retained, the bound organism can be used as a starting point for further studies.

5. Isolation of the body

According to one embodiment of the invention, the method may include isolating an organism (genotype) for further analysis. This analysis may include reproducing an organism and isolating a polynucleotide from it. The isolated phage are reproduced by conventional methods. For example, the retained phage can be brought into contact with a vector of biological amplification, for example E. coli, plus culture medium, for growing organisms for subsequent analysis. Single clones can then be tested for their ability to bind analyte retained on a substrate.

6. Sequencing of the nucleotide sequence encoding a polypeptide agent

Sequencing the nucleotide sequence encoding a polypeptide agent of a retained organism provides information for obtaining a polypeptide agent. Sequencing may include isolating an organism from an adsorbent, reproducing it, isolating a polynucleotide from it, and sequencing a nucleotide sequence in any way possible. According to another embodiment of the invention, the organism can be reproduced in situ by contacting the substrate with appropriate materials, such as cells to be infected by the organism. In another embodiment, sequencing is performed in situ. The method may include lysing the body and amplifying the nucleotide sequence in any known manner, e.g. PCR Several different organisms can be associated with various epitopes present on the surface. In this case, the elution conditions can be changed so that only one type of phage binds to the epitope.

7. Obtaining a polypeptide agent

The next important step involves obtaining a polypeptide agent. The isolated agent can be used, for example, as an adsorbent for specific target detection in diagnostics or for studying ligand / receptor interactions.

In one approach, the preparation of a polypeptide involves the initial sequencing of a nucleotide sequence encoding a polypeptide. Amino acid sequence can be deduced from the nucleotide sequence. Sequencing can be performed as described above. The sequence may serve as the basis for recombinant or chemical synthesis of the polypeptide agent.

In another embodiment, the polypeptide can be obtained by reproducing the organism. This is especially effective when the body contains many copies of the polypeptide agent. The organism can be reproduced in situ or after isolation.

The method of recombinant production of the polypeptide can be carried out as follows. The nucleotide sequence encoding the polypeptide is sequenced or isolated in any way. Then the nucleotide sequence is introduced into the expression vector. The expression vector contains an expression control sequence that is operably linked to a nucleotide sequence encoding a polypeptide. The expression vector can then be used for recombinant expression of the polypeptide agent by methods known in the art.

It is understood that a target may contain more than one epitope. Accordingly, more than one target-specific polypeptide agent can be obtained by this method.

Target-specific agents can be used as adsorbents in the preparation of probes used in clinical diagnosis or screening of potential drugs. That is, since such probes carry agents on their surface that specifically bind the target, they can be used to isolate the target from complex mixtures, such as biological samples, and to detect the target by desorption spectrometry. In addition, since the interaction between the agent and the target can be biospecific, with it a higher affinity between the two components is possible than in the case of the adsorbent obtained by the sequential resolution method described above.

8. Isolation of peptide epitopes on the target

One of the variants of this method allows peptide epitopes to be isolated on the target analyte. The method uses the "anti-idiotypic" approach. Briefly, epitopes of the target analyte are screened, for example, using a phage library. An isolated phage contains, for example, single chain antibodies that recognize epitopes on the analyte. This phage, in turn, is used to screen a second library. A phage from a second library that binds single chain antibodies from the first library contains polypeptides whose structure mimics the structure of an epitope recognized by single chain antibodies.

According to one embodiment of this method, the nucleotide sequence encoding the polypeptide agent that binds the target analyte is used to produce M13 phage, in which the agent is presented as a fusion protein with gene VIII. Thus, this phage has a shell on which hundreds of copies of the target peptide are represented. This phage is then associated with an adsorbent. Binding can be accomplished by, for example, a ligand that binds gene III, or gene III can be modified by introducing a ligand receptor on a substrate. Then the phage are brought into contact with the second library. Organisms present in the library that bind the attached phage are detected and isolated as described. Preferably, the second library contains any label so that the VIII gene protein in the library is different from the phage VIII gene protein held on the substrate. Thus, identifying a substance as a “target analyte” or as an adsorbent may depend on whether the used bound substance can sequentially bind another substance. As you can see, the ability of a bound substance to bind another substance can be reproduced and serve to identify conditions in which a permanently bound substance is selectively removed.

EXAMPLES

The following examples are provided for illustrative purposes only and do not limit the scope of the invention.

The following products and terms are used in the examples. Egg white lysozyme (1 μl, diluted with water to a concentration of 10 picomoles / μl), available from Sigma Chemical Company, St. Louis, MO. The term "human serum" refers to the composition of 1 μl of human serum diluted 1 to 5 in 20 mm sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

In this context, "mg" means milligram (s); "ml" means milliliter (s); "μl" means microliter (s); "cm" means centimer (s); "nm" means nanometer (s); "M" means molar; "mm" means millimolar; "min" means minute (s); “%” or “percent” means weight percent, unless otherwise indicated; "NaCl" means sodium chloride; "TFA" means trifluoroacetic acid.

1. RETENTANT CHROMATOGRAPHY PROTOCOLS

The following protocols are examples of procedures used in the implementation of retentate chromatography.

A. Protocol retentate mapping (using sequential chromatographic matrices)

1. Sample processing

Dilute the biological sample (i.e. serum, urine, cell extract or cultured cell medium) in 0.01% Triton X100 in HEPES or 20 mM Na-phosphate, pH 7.2. If necessary, clarify the sample by centrifugation.

2. Application of the sample

Apply a sample (1-5 μl) to a spot of anionic, nominal phase or TED-Cu (II) adsorbent. In the case of a hydrophobic adsorbent, pre-wet each spot with 0.5 μl of acetonitrile containing 0.5% TFA. Apply the sample to the stain until the acetonitrile dries. Allow the sample to concentrate on the spot (almost to dryness).

3. Flushing

a. Anionic adsorbent matrix Wash stain with 1 20 mM HEPES or Na-phosphate, pH 7.2. Add the first 2 μl of the wash solution to the stain before the sample is completely dry.

Leave the wash solution on the stain for at least 15 seconds.

Pipette 10 times. Completely remove the first wash solution, repeat the wash with a second portion (2 μl) of the solution.

Wash stain 2 with 0.2 M Nacl in 20 mM Na-phosphate, pH 7.2, as described above.

Wash the spot with 3 1 M NaCl in 20 mM Na-phosphate, pH 7.2, as described above.

Wash stain 4 with 20 mM Tris HCl, pH 8.5, as described above.

Wash stain 5 with 0.1 M Na acetate, pH 4.5, as described above.

Wash the stain 6 with 0.05% Triton X100 in 20 mM HEPES or Na-phosphate, pH 7.2, as described above.

Wash the stain with 7 3 M urea in 20 mM HEPES or Na-phosphate, pH 7.2, as described above.

Wash stain 8 with 10% acetonitrile in water as described above.

Rinse the entire matrix thoroughly with water.

Dry the chip in air.

Add 0.3 μl of the energy-absorbing molecule (saturated solution obtained in 50% acetonitrile, 0.5% trifluoroacetic acid).

Dry the chip in air.

Analyze the retained protein in each spot using a laser desorption-ionization mass spectrometer.

6. Matrix with normal phase adsorbent

Wash the spot with 1 5 mM HEPES, pH 7.0. Add the first 2 μl of the wash solution to the stain before the sample is completely dry. Leave the wash solution on the stain for at least 15 seconds. Pipette 10 times. Completely remove the first wash solution, repeat the wash with a second portion (2 μl) of the solution.

Wash stain 2 with 20 mM Na-phosphate, 0.15 M NaCl, pH 7.2, as described above.

Wash stain 3 with 20 mM Na-phosphate, 0.5 M NaCl, pH 7.2, as described above.

Wash stain 4 with 0.1 M Na-acetate, pH 4.0, as described above.

Wash the spot 5 with 0.05% Triton X100 in 20 mM Na-phosphate, 0.15 M NaCl, pH 7.2, as described above.

Wash the stain with 6 3 M urea in 20 mM Na-phosphate, 0.15 M NaCl, pH 7.2, as described above.

Rinse the stain 7 1% TFA as above.

Wash stain 8 with 30% isopropanol: acetonitrile in water as described above.

Rinse the entire matrix thoroughly with water.

Dry the chip in air.

Add 0.3 μl of the energy-absorbing molecule (saturated solution obtained in 50% acetonitrile, 0.5% trifluoroacetic acid).

Dry the chip in air.

Analyze the retained protein in each spot using a laser desorption-ionization mass spectrometer.

in. TED-Cu (II) Adsorbent Matrix

Wash the spot with 1 20 mM HEPES, 0.5 M NaCl, pH 7.2. Add the first 2 μl of the wash solution to the stain before the sample is completely dry.

Leave the wash solution on the stain for at least 15 seconds.

Pipette 10 times. Completely remove the first wash solution, repeat the wash with a second portion (2 μl) of the solution.

Wash stain 2 with 20 mM imidazole in 20 mM Na-phosphate, 0.5 M NaCl, pH 7.2, as described above.

Wash the stain with 3 100 mM imidazole in 20 mM Na-phosphate, 0.5 M NaCl, pH 7.2, as described above.

Wash the stain 4 with 0.1 M Na-acetate, 0.5 M NaCl, pH 4.0 as described above.

Wash the spot 5 with 0.05% Triton X100 in 20 mM Na-phosphate, 0.15 M NaCl, pH 7.2, as described above.

Wash the spot with 6 3 M urea in 20 mM Na-phosphate, 0.15 M NaCl, pH 7.2, as described above.

Rinse the stain 7 1% TFA as above.

Wash stain 8 with 10% acetonitrile in water as described above. Rinse the entire matrix thoroughly with water. Dry the chip in air.

Add 0.3 μl of the energy-absorbing molecule (saturated solution obtained in 50% acetonitrile, 0.5% trifluoroacetic acid).

Dry the chip in air.

Analyze the retained protein in each spot using a laser desorption-ionization mass spectrometer.

Hydrophobic adsorbent matrix

Wash the stain with 15% acetonitrile in 0.1% TFA. Add the first 2 μl of the wash solution to the stain before the sample is completely dry. Leave the wash solution on the stain for at least 15 seconds. Pipette 10 times. Completely remove the first wash solution, repeat the wash with a second portion (2 μl) of the solution.

Wash stain 2 with 50% acetonitrile in 0.1% TFA as described above.

Wash the stain 3 with 0.05% Triton X100 in 20 mM Na-phosphate, 0.15 M NaCl, pH 7.2, as described above.

Wash the stain with 4 3 M urea in 20 mM Na-phosphate, 0.15 M Nad, pH 7.2, as described above.

Rinse the entire matrix thoroughly with water.

Dry the chip in air.

Add 0.3 μl of the energy-absorbing molecule (saturated solution obtained in 50% acetonitrile, 0.5% trifluoroacetic acid).

Dry the chip in air.

Analyze the retained protein in each spot using a laser desorption-ionization mass spectrometer.

B. Protocol test antibody-antigen; receptor ligand assay (using a preactivated adsorbent matrix)

1. Immobilization of the antibody on a preactivated adsorbent matrix

Place the preactivated adsorbent matrix on a clean, flat surface. Apply antibody, or receptor, or control solution to each spot of the preactivated adsorbent matrix, pre-wetted with 0.5 μl of isopropanol (add 1 μl of antibody to the spot before the isopropanol dries).

Incubate (at 4 ° C or room temperature, 2-18 hours) in a humid chamber.

Pipette the remaining stain on the stain.

Block the remaining active sites in the spots by adding 1 ml of 1 M ethanolamine, pH 7.4 in the FBI for the whole chip and incubate in a humid chamber (at room temperature for 30 minutes).

Wash 0.5 M NaCl in 0.1 M sodium acetate, pH 4.0, as described above.

Wash 0.5 M NaCl in 0.1 M Tris-HCl, pH 8.0 as described above.

Rinse the FBI as described above. Then wrap the chip in the FBI and store at 4 ° C until ready to use.

2. Binding of antigen or ligand

Gently shake or blot the FBI off the chip.

Add 1-5 μl of sample to each spot. For samples with low antigen or ligand concentrations, place the adsorbent matrix in a bioprocessor. Rinse the spots on the chip and bioprocessor cells with 200 μl FBI twice. Add 300 μl of sample to each well. Cover with sticky tape. Incubate with shaking (at 4 ° C or room temperature, 1-18 hours).

3 Flushing

Remove the stain from the spots, wash each spot twice with 2 μl) 0.1% Triton X100 in the FBI, pH 7.2. Apply the first 2 μl of the wash solution to the stain. Allow the solution to stain for at least 15 seconds. Pipette 10 times. Completely remove the first wash solution, repeat washing with 2 μl of the second wash solution. Then followed by washing with 0.5 M NaCl in 0.1 M HEPES, pH 7.4. Rinse the entire matrix thoroughly with water.

4. Retained Protein Analysis

Dry the chip in air.

Add 0.3 μl of the energy-absorbing molecule (saturated solution of synapinic acid or EAM1 or CHSA prepared in 50% acetonitrile, 0.5% three fluoroacetic acid).

Dry the chip in air.

Analyze the retained protein in each spot using a laser desorption-ionization mass spectrometer.

II. LYSOZIM RECOGNITION PROFILE

We obtained a lysozyme recognition profile using high resolution retentate chromatography. The profile includes the resolution of lysozyme on six adsorbents with various selectivity threshold modifiers. The result is presented in the form of 40 different spectrograms that differentially characterize the physicochemical properties of lysozyme.

A. Lysozyme recognition profile using a hydrophilic adsorbent matrix

Egg white lysozyme is added to various spots of a chromatographic adsorbent matrix deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, pH 7.

(2) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(3) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(4) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

(5) 1% TFA

(6) 10% acetonitrile in water,

(7) 20% acetonitrile in water,

(8) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and

(9) 3M urea in 20 mM sodium phosphate buffer, pH 7.0

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a mass spectrometer using a nitrogen laser (355 nm) and a 60 cm long sleeve. Data was analyzed by computer and transferred to GRAMS / 32c (available from Galactic Industries Corporation) for subsequent presentation.

Fig. 5A shows a composite mass spectrum for a lysozyme recognition profile on a normal phase chromatographic adsorbent matrix. The lower profile represents the intensity signals of lysozyme retained on an adsorbent with silicon oxide after washing only with a buffer with pH 7.0. The addition of sodium chloride (0.2-0.4 M) as a modifier of the selectivity threshold reduces lysozyme retention. This indicates the fact that the interaction of lysozyme (the main protein) with an adsorbent - silicon oxide (negatively charged at pH 7) occurs by the ion-exchange mechanism. A decrease in the pH of the selectivity threshold modifier, for example, to pH 4.5 in a sodium acetate buffer, or <2 in 1% TFA, almost completely removes the negative charge from the silicon oxide adsorbent and lysozyme does not linger on it. The introduction of polarity modulating agents (i.e., organic solvents (e.g., acetonitrile), or detergent (Tween 20), or urea as a modifier of the selectivity threshold also reduces the interaction of lysozyme with a silicon oxide adsorbent.

B. Lysozyme recognition profile using a hydrophobic adsorbent matrix

Egg white lysozyme is added to various spots of a chromatographic adsorbent matrix deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 0.1% TFU

(2) 10% acetonitrile in 0.1% TFA,

(3) 20% acetonitrile in 0.1% TFA,

(4) 50% acetonitrile in 0.1% TFA,

(5) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and

(6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a mass spectrometer using a nitrogen laser (355 nm) and a sleeve 60 cm long. Data was analyzed using a computer and transferred to GRAMS / 32c for subsequent presentation.

5B contains the composite mass spectrum of lysozyme recognition profile on a chromatography matrix with a hydrophobic adsorber C _s. The lower profile represents the intensity signals of lysozyme retained on an adsorbent with hydrophobic C3 after washing with only 0.1% TFA. The use of an agent that simulates polarity (i.e., acetonitrile) as a modifier of the selectivity threshold reduces lysozyme retention on a hydrophobic C ₃ adsorbent. The concentration of acetonitrile used to elute lysozyme from a hydrophobic C ₃ adsorbent was varied in the range of 20-50%. The introduction of detergent (Tween 20) or urea as a modifier of the selectivity threshold slightly reduces the retention of lysozyme on a hydrophobic C ₃ adsorbent.

B. Lysozyme recognition profile using a phenyl hydrophobic adsorbent matrix

Egg white discozyme is added to various spots of the chromatographic adsorbent matrix with a polypropylene (C3-hydrophobic) adsorbent deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 0.1% TFU

(2) 10% acetonitrile in 0.1% TFA,

(3) 20% acetonitrile in 0.1% TFA,

(4) 50% acetonitrile in 0.1% TFA,

(5) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and

(6) 3M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a mass spectrometer using a nitrogen laser (355 nm) and a sleeve 60 cm long. Data was analyzed using a computer and transferred to GRAMS / 32c for subsequent presentation.

On figs presents a composite mass spectrum for the recognition profile of lysozyme on a chromatographic adsorbent matrix with hydrophobic phenyl. The lower profile represents the intensity signals of lysozyme retained on an adsorbent with hydrophobic phenyl after washing with only 0.1% TFA. The use of an agent that simulates polarity (i.e., acetonitrile) as a modifier of the selectivity threshold reduces lysozyme retention. The concentrations of acetonitrile used to elute lysozyme from a hydrophobic C ₃ adsorbent varied between 20-50%, however, when the intensities of the peaks of lysozyme held on C ₃ and phenyl were compared under the same conditions for washing with acetonitrile (20%), then the interaction of lysozyme with phenyl adsorbent was less strong. Using a detergent (i.e., Tween 20) or urea as a selectivity threshold modifier also significantly reduces lysozyme retention on hydrophobic phenyl adsorbent.

D. Lysozyme recognition profile using an anionic adsorbent matrix

Egg white lysozyme is added to various spots of the anionic matrix (SO ₃ ^- ) adsorbent matrix (i.e., cation exchange adsorbent) deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, pH 7.0.

(2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(4) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(5) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

(6) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and

(7) 3M urea in 20 mM sodium phosphate buffer, pH 7.0

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a mass spectrometer using a nitrogen laser (355 nm) and a sleeve 60 cm long. Data was analyzed by computer and transferred to GRAMS / 32c for subsequent presentation.

On fig.5D presents a composite mass spectrum for the recognition of lysozyme on a cation exchange chromatographic adsorbent matrix. The lower profile represents the intensity signals of lysozyme retained on the anionic adsorbent after washing only with pH 7.0 buffer. The introduction of increasing concentrations of sodium chloride (0.2-0.4 M) as a modifier of the selectivity threshold reduces lysozyme retention. This indicates the fact that the interaction of lysozyme (the main protein) with the anionic adsorbent occurs by the ion-exchange mechanism. Elution of lysozyme required a NaCl concentration of -0.4 M. Decreasing the pH of the selectivity threshold modifier, for example, to pH 4.5 in sodium acetate buffer, did not affect lysozyme retention by a strong anionic adsorbent. The introduction of polarity modulating agents (i.e. detergents such as Tween 20, or urea as a modifier of the selectivity threshold) reduces the interaction of lysozyme with an anionic adsorbent. This indicates that the interaction of the hydrophobic protein - lysozyme with anionic adsorbent is modulated by the eluant polarity.

D. Recognition profile of lysozyme using a cationic adsorbent matrix

Egg white lysozyme is added to various spots of the adsorbent matrix with a cationic adsorbent (quaternary amine) deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, pH 7.0.

(2) 0.1 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(3) 0.2 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(4) 0.4 M NaCl in 20 mM sodium phosphate buffer, pH 7.0,

(5) 25 mM sodium acetate buffer, 0.125 M NaCl, pH 4.5,

(6) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, or

(7) 3M urea in 20 mM sodium phosphate buffer, pH 7.0

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a mass spectrometer using a nitrogen laser (355 nm) and a sleeve 60 cm long. Data was analyzed by computer and transferred to GRAMS / 32c for subsequent presentation.

On fige presents a composite mass spectrum for the recognition profile of lysozyme on a chromatographic adsorbent matrix with a cationic (anion exchange) adsorbent. The retention of the main lysozyme protein on the cationic adsorbent is very small. The effect of modulating selectivity threshold modifiers on lysozyme retention is minimal.

E. Recognition profile of lysozyme using an absorbent matrix with immobilized metal ions

Egg white lysozyme is added to various spots of an absorbent matrix with immobilized metal ions (iminodiacetate-Cu) deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

(2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

(3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

(4) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0 or

(5) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a mass spectrometer using a nitrogen laser (355 nm) and a sleeve 60 cm long. Data was analyzed using a computer and transferred to GRAMS / 32c for subsequent presentation.

FIG. 5F shows a composite mass spectrum for a lysozyme recognition profile on an immobilized metal chromatographic adsorbent matrix. The lower profile represents the intensity signals of lysozyme retained on an adsorbent with immobilized copper after washing only with pH 7.0 buffer. The use of a histidine-binding competitive affinity ligand (i.e., imidazole) as a selectivity threshold modifier cancels lysozyme retention. This fact indicates that in the interaction of lysozyme (which has a single histidine residue in its sequence) with an adsorbent based on immobilized copper ions, the coordinate covalent binding mechanism is involved. Lowering the pH of the selectivity threshold modifier to 4.5 in sodium acetate buffer also reduces lysozyme retention on an adsorbent with immobilized copper. It is believed that this is the result of protonation of the histidine residue in lysozyme, which suppresses the covalent coordinate interaction. Using a detergent (i.e. Tween 20) does not affect the interaction. The use of urea completely cancels the retention of lysozyme on an adsorbent with immobilized copper.

III. DETECTION OF ANALYTES IN HUMAN SERUM

We have resolved human serum analytes using a variety of adsorbents and eluants. The results show that analytes are differentially retained by various adsorbents, and that retention chromatography provides information on analytes of both low and high molecular weights.

A. Profile for the recognition of human serum proteins using an absorbent matrix with immobilized metal ions

Human serum is applied to various spots of an absorbent matrix with immobilized metal ions (Tris (carboxymethyl) ethylenediamine-Cu) deposited on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each individual adsorbent spot is washed with one of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

(2) 5 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 7.0,

(4) 0.05% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0 and

(5) 3M urea in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix is analyzed on a nitrogen laser mass spectrometer (355 nm) and a 60 cm tube. The data are analyzed by computer and transferred to GRAMS / 32c for subsequent presentation.

On figa and 6B presents a composite mass spectrum for the recognition profile of serum proteins of low and high molecular weights on a chromatographic adsorbent matrix with immobilized metal. The lower profile represents serum proteins retained on an adsorbent with immobilized copper after washing only with pH 7.0 buffer. Using a histidine-binding competitive affinity ligand (i.e., imidazole) or detergent (i.e. Tween 20), or urea as a selectivity threshold modifier, or lowering the selectivity threshold modifier pH to pH 4.5 differentially enhances or decreases retention of various components of a complex mixture of proteins on a single adsorbent.

B. Profile recognition of human serum proteins using many different adsorbents

Human serum was applied to various spots of the absorbent matrix with the following various adsorbents:

(1) With ₃ hydrophobic,

(2) phenyl hydrophobic,

(3) anion exchange,

(4) cation exchange, and

(5) immobilized metal (tris (carboxymethyl) ethylenediamine-Cu).

Each adsorbent is coated on a silicon oxide coated stainless steel substrate. After incubation in a humid chamber at room temperature for 15 minutes, each spot of the adsorbent is washed with 0.05% Tween 20 in 20 mM phosphate buffer, 0.15 M NCl, pH 7.0 as a selectivity threshold modifier.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a nitrogen laser mass spectrometer (355 nm) and a 60 cm tube. Data was analyzed by computer and transferred to GRAMS / 32c for subsequent presentation.

On figa and 7B presents a composite mass spectrum for the recognition profile of serum proteins on various adsorbents of chromatographic adsorption matrices. The use of a single selectivity modifier with many different adsorbents (each of which has different binding characteristics) differentially enhances or decreases the retention of various components of a complex protein mixture on different adsorbents.

IV. DETECTION OF ANALYTES IN THE URINE OF PREMATURE CHILDREN

A. Resolution of analytes in the urine of a premature baby using various adsorbents indirectly and one eluant (water)

The urine of a premature baby (2 μl) is applied to various spots of a carbonated PEEK polymer substrate coated with the following various adsorbents:

(1) C ₈ hydrophobic (Octyl Sepharose, available from Sigma)

(2) phenyl hydrophobic (Phenyl Sepharose, available from Sigma)

(3) anion exchange (Q-Sepharose, available from Sigma)

(4) cation exchange (S-Sepharose, available from Sigma)

(5) immobilized metal (IDA-Cu, Chelating Sepharose, available from Pharmacia)

(6) immobilized metal (Tris (carboxymethyl) ethylenediamine-Cu Sepharose).

After incubation in a humid chamber at room temperature for 15 minutes, each spot of the adsorbent is washed with water as a selectivity threshold modifier. Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed with 1 μl of water twice. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix is analyzed on a desorption-ionization mass spectrometer (Hewlett Packard, Model 2030) with a nitrogen laser (355 nm) and a 150 cm tube. The data are analyzed using the HP MALDI TOF computer program and transferred to GRAMS / 32c for subsequent presentation.

On figa and 8B presents a composite mass spectrum for the recognition profile of proteins of low and high molecular weights contained in the urine of a premature baby, on various adsorbents of chromatographic adsorption matrices. The use of a single selectivity modifier (i.e., water) with different adsorbents (each of which has different binding characteristics) differentially enhances or decreases the retention of various components

B. Resolution of analytes in the urine of a premature baby using phenyl-hydrophobic adsorbent indirectly bound to the substrate and three different eluants

The urine of a premature baby (2 μl) is applied to various spots of a carbonized PEEK polymer substrate coated with a phenyl hydrophobic adsorbent (Phenyl Sepharose, available from Sigma). After incubation in a humid chamber at room temperature for 15 minutes, each spot of the adsorbent is washed with one of the following eluants (selectivity threshold modifiers):

(1) water

(2) 2 M urea in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0, and

(3) 0.1% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCI, pH 7.0.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix is analyzed on a desorption-ionization mass spectrometer (Hewlett Packard, Model 2030) with a nitrogen laser (355 nm) and a 150 cm tube. The data are analyzed using the HP MALDI TOF computer program and transferred to GRAMS / 32c for subsequent presentation.

Figure 9 presents a composite mass spectrum of the recognition profile of the urine of a premature baby on a hydrophobic phenyl adsorbent chromatographic matrices. The use of different eluants with different elution characteristics with one adsorbent differentially enhances or weakens the retention of various components of a complex protein mixture. One of the components (indicated by “*”) was selectively retained on a hydrophobic phenyl adsorbent when 0.1% Tween 20 was used as eluant in the FBI.

V. IDENTIFICATION OF PROTEINS IN THE CULTURAL ENVIRONMENT OF TWO DIFFERENT CELL LINES

This example illustrates the identification using chromatographic adsorbent matrices of proteins that are differentially expressed in cells.

Cells of two different lines of breast cancer were cultured for one period of time in a culture medium of constant composition. After concentration in a filter cell, an aliquot of each culture medium with a volume of 1 μl was applied to various spots of an adsorbent matrix (Ciphergen Biosystems, Inc., Palo Alto, CA) with immobilized metal (Tris (carboxymethyl) ethylenediamine-Cu) deposited on a stainless steel substrate, coated with silica. After incubation in a humid chamber at room temperature for 15 minutes, each spot of the adsorbent is washed with one of the following eluants (selectivity threshold modifiers):

(1) 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0.

(2) 20 mM imidazole in 20 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0,

(3) 0.1 M sodium acetate buffer, 0.5 M NaCl, pH 4.5,

(4) 0.1% Tween 20 in 20 mM sodium phosphate buffer, 0.15 M NaCl, pH 7.0.

(5) 3M urea in 10 mM sodium phosphate buffer,

(6) 1% TFA.

Each washing includes tripling 1 μl of the washing solution on an adsorbent spot. This process is repeated with a fresh aliquot of the wash solution. Then the spot of the adsorbent is washed twice with 1 μl of water. 0.3 μl aliquot of synapinic acid (5 mg / ml 50% acetonitrile: 0.5% TFA) was added and dried in air. The matrix was analyzed on a nitrogen laser mass spectrometer (355 nm) and a 60 cm tube. Data was analyzed by computer and transferred to GRAMS / 32c (Galactic Industries Corporation) for subsequent presentation.

On figa presents a composite mass spectrum of the recognition profile for proteins secreted by cell line 1 on chromatographic adsorption matrices with immobilized metal (Cu). The use of different eluants on the same adsorbent with different thresholds of selectivity differentially enhances or weakens the retention of various components of such a complex protein mixture as the cell culture medium.

On figv presents a composite mass spectrum of the recognition profile for proteins secreted by both cell lines on chromatographic adsorption matrices with immobilized metal (Cu). The same eluant 1.0% Tween 20 + 3 M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0 was used to wash unbound material. The peak designated 7532 Da is a major retained protein from a set of proteins secreted by cell line 1 that is not expressed in cell line 2.

On figs presents a composite mass spectrum of the recognition profile for proteins secreted by cell line 2 on chromatographic adsorption matrices with immobilized metal (Ni). When using the same eluant, 1.0% Tween 20 + 3 M urea in 10 mM sodium phosphate buffer, 0.5 M NaCl, pH 7.0, but adsorbents with different surface interaction potentials (i.e., immobilized Ni versus immobilized Cu) peak 7532 Yes represents the only protein retained among the proteins secreted by cell line 1. The box shows the same mass spectrum on an enlarged scale. The smaller peak 3766 Da represents the same protein, but with a double charge.

Figure 10D shows a composite mass spectrum of the recognition profile for proteins secreted by cell line 1 on immobilized metal (Ni) chromatographic adsorption matrices before (lower profile) and after (upper profile) in trypsin treatment. The peptide map obtained for pure protein is a fingerprint of this protein and can be used for identification.

VI. COMPARISON OF RETENTANT CHROMATOGRAPHY WITH TWO-DIMENSIONAL GEL ELECTROPHORESIS

One of the advantages of retentate chromatography is the ability to quickly resolve analytes in various measurements, which provides high density information about various physicochemical characteristics. Conversely, two-dimensional gel electrophoresis provides resolution in only two dimensions.

Figure 11 shows the recognition profile of the urine of a premature baby on a hydrophobic phenyl adsorbent chromatographic matrices. The use of various eluants and adsorbents gives multiparameter information. Using different conditions of selectivity differentially enhances or weakens the retention of various components of a complex protein mixture (such as urine of a premature baby), which leads to a detailed resolution of the analytes.

On the contrary, FIG. 12 shows the two-dimensional resolution of the proteins contained in the urine of a premature baby in pI and molecular weight. The gel provides information on only two parameters compared to six parameters of the adsorbents used for retentate chromatography. Spots are not as solvable as in mass spectrometry and resolution in the regions of very high and very low molecular weights is limited.

VII. SEQUENTIAL EXTRACTION OF ANALITES FROM SAMPLE

The analytes can be sequentially extracted from the sample, sequentially processing the sample under various conditions of selectivity, followed by collection of the undue sample.

Hemophilus knockout mutant lysate is prepared in 10% glycerol, 50 mM EDTA. After centrifugation, the supernatant was diluted 1: 3 in 0.01% Triton X100, 25 mM HEPES, pH 7.4. An aliquot (2 μl) of the diluted sample is applied to the anionic site of the adsorbent matrix. After incubation at room temperature for 30 minutes, the material remaining at the anionic site is transferred to the normal phase site of the adsorbent matrix. The anion site spot is washed twice with 2 μl of 0.01% Triton X100, 25 mM HEPES. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot. The washings are mixed with the sample initially carried on the normal phase site.

After incubation at room temperature for 30 minutes, the material remaining at the normal phase site is transferred to the Ni (II) site of the adsorption matrix. The stain of the normal phase site is washed twice with 2 μl of phosphate buffered saline. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot. The washings are mixed with the sample originally carried on a Ni (II) stain.

After the sample was concentrated almost dry on a Ni (II) spot, unbound analytes were isolated by washing twice with 2 μl of 10 mM imidazole in phosphate buffered saline. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot.

Washings are transferred to the stain of the aliphatic hydrophobic site of the adsorbent matrix.

The sample is allowed to concentrate almost dry on a hydrophobic site, unbound analytes are removed by washing twice with 2 μl of 5% acetonitrile in 0.1% TFA. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot.

Each spot of anionic, normal phase, Ni (II) and hydrophobic site is washed with 2 μl of water to remove the remaining buffer. A 0.3 μl aliquot of a solution of synapinic acid in 50% acetonitrile is added, 0.5% TFA is added to each stain. The analytes retained at each site are analyzed on a laser desorption-ionization mass spectrometer.

On figa-190 shows the retention map of the lysate of Hemophilus on an absorbent matrix. On the adsorbents, numerous peaks are observed in the mass range from 3000 to 25000 Da. Note that each adsorbent exhibits a different retention of each analyte present in the sample.

Viii. SEQUENTIAL RESOLUTION OF ANALYT

By adding new binding or elution characteristics to the selectivity condition that the analyte allows, a selectivity condition that provides improved analyte resolution can be obtained. In this example, the sample is bound on a Cu (II) adsorbent and treated with the first eluant and then two second eluants. The second eluants differ from the first by the addition of an excellent elution condition. Each added condition improves analyte resolution.

The stationary-phase Hemophilus lysate, taken in 10% glycerol, is diluted 1: 120 mM sodium phosphate, 0.5 M sodium chloride, pH 7.0. After centrifugation, an aliquot of the supernatant with a volume of 150 μl was incubated with spots of the Cu (II) site of the adsorbent matrix in a bioprocessor. After stirring in the cold for 30 minutes, the sample was removed. Each stain is washed with a different eluant. The first spot is washed with 150 μl of 20 mm sodium phosphate, 0.5 M sodium chloride, pH 7.0. The second spot is washed with 150 μl of 0.05% Triton X100 with the addition of 0.15 M NaCl, pH 7.0. The third spot is washed with 150 μl of 100 mm imidazole with the addition of 20 mm sodium phosphate, 0.15 M NaCl, pH 7.0. Each wash involves incubating the wash solution on the spot for 5 minutes with stirring. Washing is repeated twice. Then, each spot is washed with water to remove detergent and buffer.

The absorbent matrix is removed from the bioprocessor. To each stain, add a 0.3 μl aliquot of synapinic acid in 50% acetonitrile, 0.5% TFA. The analytes held on each spot are analyzed using a deletion-ionization mass spectrometer.

On figa-20C presents a retention map of the lysate of Hemophilus on the site Cu (II) of the adsorbent matrix after washing under the three elution conditions described above. Numerous peaks are observed in the mass range from 2000 to 18000 Da. The protein indicated by “*” is the only minor component on the retention card of FIG. 20A. When the selectivity condition is modified by adding a detergent, Triton X100 (Fig. 20B) to the same buffer, the * protein is better retained than other analytes, and therefore resolves better. When the selectivity condition is modified by adding to the same buffer an affinity substitute, imidazole (FIG. 20C), the protein “*” is highly resolved from other analytes on the retention map.

This strategy of sequentially identifying selectivity conditions with improved analyte resolution can be adapted to develop a method for the preparative purification of this protein from the total Hemophilus lysate.

IX. DIFFERENTIAL ANALYT EXPRESSION: DETECTION OF MARKER PROTEIN

A. Human Serum

Aliquots of serum 0.5 μl, taken from healthy and sick people, are diluted with an equal volume of 20 mm sodium phosphate, 0.5 M NaCl, pH 7.0. Each sample is applied to a separate spot of the Cu (II) site of the adsorbent matrix. After incubation at 4 ° C for 1 hour, each spot is washed twice with 2 μl of 20 mM sodium phosphate, 0.5 M NaCl, pH 7.0 two times. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot. Finally, each spot is washed with 2 μl of water to remove the remaining buffer. To each stain, add a 0.3 μl aliquot of synapinic acid in 50% acetonitrile, 0.5% TFA. The analytes held on each spot are analyzed using a deletion-ionization mass spectrometer.

The protein indicated by “*” in FIG. 21D is present in significantly higher amounts in the serum of the patient than in the serum of a healthy person. The results indicate that the method of detecting disease markers can be used for clinical diagnosis.

B. Urine of the mouse

Aliquots (1 μl) of urine taken from normal, diseased and drug-treated mice are applied to the spots of the Cu (II) site of the adsorbent matrix. After incubation at room temperature for 10 minutes, each spot is washed with 2 μl of 100 mm imidazole in 20 mm sodium phosphate, 0.15 M NaCl, pH 7.0 twice. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot. Finally, each spot is washed with 2 μl of water to remove the remaining buffer. To each stain, add a 0.3 μl aliquot of synapinic acid in 50% acetonitrile, 0.5% TFA. The analytes held on each spot are analyzed using a deletion-ionization mass spectrometer.

Figure 1 shows the retention maps of normal urine (control), sick and drug-treated mice. One of the analytes was present in the urine of the sick mouse in significantly higher quantities (middle panel), the same analyte was not found in the urine of a normal mouse (upper panel) and was found in significantly lower amounts in the urine of the mouse treated with the drug (lower panel). This analyte can be used as a potential marker of the disease. To illustrate the reliability of the quantitative diagnostic test, the peak area of the retained marker protein was calculated, which is shown in the table. Clear quantitative differences are observed between the urine samples of the patient and the treated mice. To compensate for the experimental deviations, an internal standard analyte was used. The normalized peak area of the disease marker (i.e., the peak area of the marker divided by the peak area of the internal standard) for each urine sample is presented on the bottom panel. There is at least a ten-fold decrease in the marker of the disease in the urine after treatment with the drug.

B. Human urine

Urine samples from normal people and cancer patients were diluted 1: 2 in 1.01% Triton X100 in phosphate buffered saline. Aliquots of the urine of healthy or sick people with a volume of 1.5 μl were applied to various spots of the aliphatic hydrophobic site of the adsorbent matrix moistened with 0.5 μl of a mixture of isopropanol / acetonitrile (1: 2), 0.1% TFA. After incubation at 4 ° C for 30 minutes, each spot is washed twice with 2 μl of 50% ethylene glycol in 10 mM Tris HCl, 0.05 M NaCl, pH 7.5. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot. Finally, each stain is washed with 2 μl of water to remove the remaining ethylene glycol and buffer. To each stain, add a 0.3 μl aliquot of synapinic acid in 50% acetonitrile, 0.5% TFA. The analytes held on each spot are analyzed using a deletion-ionization mass spectrometer.

Retention maps of urine samples from four cancer patients and a healthy person are shown in FIG. 23A. After washing with 50% ethylene glycol in the Tris / NaCl buffer, numerous protein peaks were retained at the hydrophobic site of the adsorbent matrix. To identify possible disease markers, differential maps were constructed for urine samples of individual patients and normal urine samples. Each line in the difference diagram, which exceeds the average value, represents an analyte, which is present in increased amounts in the urine of patients. (Fig.23B-23D). Variations in the nature of the differential maps reflect individual fluctuations in the population. However, the analyte in the 5000 Da region (indicated by “*”) and the analyte cluster in the 7500 Da region (indicated by “*”) were constantly present in increased amounts in all patients and therefore can be identified as possible markers of the disease.

X. RETAINING A PHAGE FROM A PHAGE LIBRARY

Viruses adsorbed on the surface of a protein chip can be detected by desorption spectrometry. Antibodies to viral surface proteins used as adsorbents can retain viruses. A target protein used as an adsorbent can retain a phage carrying a single chain antibody to a target.

A. Determination of the sensitivity of the antibody using an absorbent substrate

Phage M13 (10 ¹² particles / ml) in a growth medium was sequentially diluted with 0.01% Triton X100 in 25 mM HEPES, pH 7.4. Aliquots (0.25 μl) of each dilution of the phage suspension are applied to the spots of the aliphatic hydrophobic site of the adsorbing matrix. Aliquots (0.3 μl) of SNA in 50% acetonitrile, 0.5% TFA are then added. Samples were analyzed by laser desorption-ionization mass spectrometry.

Protein of gene VIII of phage M13 was detected on a matrix with high sensitivity. figa-24E. A detectable signal (signal-to-noise> 2) was obtained by diluting the phage suspension 10,000,000 times.

B. Identification of M13 phage on an adsorbent matrix

Rabbit anti-M13 antibodies (Stratagene) were immobilized on Protein A Hyper D (BioSepra) and thoroughly washed with phosphate buffered saline, pH 7.0. An aliquot of 1-10 μl of a suspension of phage M13 (10 ¹² particles / ml) in growth medium was incubated with 1 μl of an aliquot of immobilized anti-M13 antibodies at 4 ° C overnight. After washing with 0.05% Tween 20 in a phosphate buffer solution, pH 7.0, and then with water to remove detergent and buffer, an aliquot of the retained phage was analyzed by laser desorption ionization spectrometry in the presence of synapinic acid.

The anti-M13 antibody control showed only the antibody signal (single and double charged). figa. When the M13 phage was retained by the antibody, the most easily identifiable protein peaks of the phage were gene III protein and gene VIII protein fused to the single chain antibody of FIG. 25B. Since the protein of gene VIII of phage M13 is detected by this method with great efficiency, it can serve as an indicator of phage retention.

B. Specific retention of phage M13 carrying a single chain antibody

The HIV-1 Tat protein (McKesson BioServices) has been coupled to a preactivated substrate. After blocking with ethanolamine, the matrix was washed with 0.005% Tween 20 in a phosphate buffer solution, pH 7-0, and then 0.1% BSA in a phosphate buffer solution, pH 7.0. Serial dilutions of M13 phage carrying a single chain anti-Tat protein antibody were incubated on an Tat protein adsorption matrix at 4 ° C. overnight. A negative control of serial dilutions of M13 phage that did not carry a single-chain antibody against Tat protein was also incubated on an adsorption matrix with Tat protein under similar conditions. The matrices were washed with 0.005% Tween 20 in phosphate buffered saline, 1 M urea in phosphate buffered saline, pH 7.0, and finally with water to remove the buffer and urea. Then add a 0.3 μl aliquot of CHSA in 50% acetonitrile, 0.5% TFA. The retained phage are analyzed using a dispersion-ionization mass spectrometer.

The specific binding of phage M13 carrying a single chain anti-Tat protein antibody was concentration dependent (bold line). figa-260. Nonspecific binding of the non-specific M13 phage on the adsorbing matrix was minimal (dashed line). These results illustrate a very sensitive phage detection method that contains a gene encoding a single chain antibody that specifically recognizes a target analyte.

Xi. RECEPTOR AND LIGAND BINDING COMPLETION SCREENING SCREENING

The method of the present invention can be used to establish that the test agent modulates ligand binding to a receptor. In this example, we show that retentate chromatography can detect inhibition of binding between TGF-b and the immobilized TGF-b receptor using the free TCP-b receptor as an adsorbent.

The recombinant receptor TGF-b-fused Fc protein (R&D, Minnesota) was specifically attached to an adsorption matrix with protein G. TGF-b (R&D, Minnesota) was sequentially diluted with conditioned cell medium (concentrated 2.5x) and incubated overnight at 4 ° C on an adsorbent matrix with receptor-Fc protein G. Another set of serial dilutions of TGF-b in a conditioned cell environment was incubated on an adsorbent matrix with receptor-Fc protein G in the presence of a modulating agent. In this case, the modulating agent was the free TGF-b receptor. After incubation under the same conditions, the chip was washed with 0.05% Triton X100 in the FBI and then 3 M urea in the FBI. A 0.3 μl aliquot of stnapinic acid was added to each spot and analyzed by a dispersion-ionization mass spectrometer.

On figa presents the specific binding of 1 μg / ml TGF-b on an adsorbing matrix with receptor-Fc protein G (bold line). There was little or no protein binding from the conditioned cell medium. On figv presents the specific binding of 100 ng / ml TGF-b on an adsorbing matrix with receptor-Fc protein G (bold line). When the incubation of TGF-b on an adsorbent matrix with receptor-Fc protein G was carried out in the presence of a modulating agent (free TGF-b receptor), the binding was completely canceled with the addition of 100 ng / ml TGF-b (Fig. 27A, dashed line) and was only observed trace binding upon addition of 1 μg / ml TGF-b (Fig. 27B, dashed line). In this example, the modulating agent (the same receptor) had a high specific binding affinity for the ligand, thereby providing the target analyte with very effective competition for binding. In other cases, the ratio of the target analyte bound to the adsorbent in the presence and absence of a modulating agent is indicative of the effectiveness of the modulating agent.

XII. RESOLVING ABILITY OF RETENTANT CHROMATOGRAPHY

This example demonstrates the ability of retentate chromatography in parallel with the processing of the sample under various conditions of selectivity to resolve proteins in the sample.

Hemophilis influenzae lysate in 10% glycerol was prepared. After centrifugation, the supernatant was diluted 1: 3 in 0.01% Triton X100 in 25 mM HEPES, pH 7.4. An aliquot of a 2 μl diluted sample is applied to the spot of the cationic site on a sorbent matrix. After incubation at room temperature for 30 minutes, the stain is washed with 25 mM HEPES, pH 7.4. A second aliquot of a 2 μl diluted sample is applied to a spot of an aliphatic hydrophobic site on a sorbent matrix. After incubation at room temperature for 30 minutes, the stain is washed with water. A third aliquot of a 2 μl diluted sample is applied to the spot of the Cu (II) site on the sorbing matrix. After incubation at room temperature for 30 minutes, the stain is washed with 0.05% Triton X100 and phosphate buffered saline, pH 7.4. Then, to each stain, add a 0.3 μl aliquot of sinapinic acid in 50% acetonitrile, 0.5% TFA. The analytes held on each site are analyzed using a deportation-ionization mass spectrometer.

The results are shown in Fig.28-31. This result illustrates the method of combinatorial separation, including parallel separation and detection of multiple analytes.

Xiii. SEQUENTIAL ASSEMBLY OF MULTIDIMENSIONAL STRUCTURES

This example illustrates a method for binding a secondary adsorbent to a primary adsorbent. The secondary adsorbent serves as a specific adsorbent for the target analyte.

An aliquot of the 0.5 μl GST fusion receptor was diluted with 20 mM Tris, 100 mM sodium chloride. 0.4% NP40 is added to the normal site of the adsorbing matrix, pH 7.2- The solution is allowed to concentrate on the spot almost until dry. The stain is washed with 2 μl of 10 mM Tris, 50 mM sodium chloride, pH 7.2 three times. Finally, the stain is washed with 2 μl of water twice to remove the remaining buffer. Then, 0.3 μl aliquot of synapinic acid in 50% acetonitrile, 0.5% TFA is added to the stain. The retained fusion GST receptor is analyzed using a deportation ionization mass spectrometer (Fig. 32).

An aliquot of the fused GST receptor with a volume of 0.5 μl in 20 mM Tris, 100 mM sodium chloride, 0.4% NP40, pH 7.2 is applied to the normal site of the adsorbing matrix. As a negative control, a sample containing only GST protein (without receptor) is applied to another spot. The solution is allowed to concentrate on the spot almost to dryness. Then, 0.5 μl of 10 mM Tris, 50 mM sodium chloride, pH 7.2 are added to each spot. The solution was pipetted after 10 seconds of incubation at room temperature.

An aliquot (1 μl) of a solution containing one specific ligand from a library of 96 other ligands is immediately added to the stain. The adsorbent matrix is incubated in a humid chamber at room temperature for 1 hour. Then each spot is washed with 2 μl of a mixture of 30% isopropanol: acetonitrile (1: 2) in water twice. Each washing includes tenfold pipetting of the washing solution on an adsorbent spot. Then, a 0.3 μl aliquot of a solution of a-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.5% TFA is added to the stain. The ligand retained at the receptor is analyzed using a deletion-ionization mass spectrometer.

On figa shows the binding of a specific ligand from a library of 96 other ligands with a fused GST receptor, retained at the normal site of the adsorbing matrix. On figv shows that there is no binding of the ligand only to the GST protein (without receptor), retained on the same matrix, which serves as a negative control of the experiment.

The present invention claims new materials and methods for retentate chromatography. While specific examples are provided, the description is illustrative and does not limit the scope of the invention. When acquainted with this description for a qualified specialist will be apparent many possible variations of the invention. Therefore, the scope of the invention is determined without reference to the description by the following claims.

Claims (34)

1. A method of obtaining a substrate for detecting at least one analyte in a sample, comprising the following steps: a) processing the sample in at least two conditions of selectivity, determined by the combination of adsorbent and eluant, which makes it possible to retain the analyte with an adsorbent; b) identification by desorption spectrometry of at least one selectivity condition under which the analyte is retained; and C) obtaining a substrate containing at least one adsorbent under the identified condition of selectivity. 2. The method according to claim 1, characterized in that the identification step comprises identifying at least one selectivity condition under which a plurality of analytes is held. 3. The method according to claim 1, characterized in that the step of obtaining the substrate involves obtaining a substrate containing many adsorbents that retain the analyte under this selectivity condition as a multiplex adsorbent. 4. A method for diagnosing a subject of a disease by detecting a plurality of diagnostic markers, comprising the steps of: a) contacting at least one adsorbent attached to a substrate with a biological sample taken from a subject such that at least one adsorbent binds to diagnostic markers; b) washing at least one adsorbent with at least one eluant to remove unbound material and ensure retention of diagnostic markers; c) detection of retained diagnostic markers by desorption spectrometry; and e) establishing a diagnosis based on the detection of multiple diagnostic markers. 5. The method according to claim 4, characterized in that the plurality of adsorbents attached to at least one substrate, with each adsorbent attached in a different predetermined location. 6. The method according to claim 4, characterized in that the diagnosed disease is cancer, heart disease, autoimmune disease, viral infection, Alzheimer's disease and diabetes. 7. The method according to claim 4, characterized in that the biological sample is selected from the group comprising tissue, blood, urine, feces, lymph, cerebrospinal fluid and intraarticular fluid. 8. The method according to claim 4, characterized in that the biological sample is obtained from a pathological cell. 9. The method according to claim 4, characterized in that at least one adsorbent is a hydrophobic adsorbent. 10. The method according to claim 4, characterized in that at least one adsorbent is a thiophilic interaction adsorbent. 11. The method according to claim 4, characterized in that at least one adsorbent is a normal phase adsorbent. 12. The method according to claim 4, characterized in that at least one adsorbent is an anionic adsorbent. 13. The method according to claim 4, characterized in that at least one adsorbent is a cationic adsorbent. 14. The method according to claim 4, characterized in that at least one adsorbent is a metallonic adsorbent. 15. The method according to claim 4, characterized in that at least one adsorbent is a glycoprotein interaction adsorbent. 16. The method according to claim 4, characterized in that at least one adsorbent contains nucleic acid. 17. The method according to claim 4, characterized in that at least one adsorbent contains many antibodies that bind to diagnostic markers. 18. The method according to 17, characterized in that the antibodies are attached to the substrate in the same predetermined locations. 19. The method according to 17, characterized in that the diagnostic markers are bound by antibodies in various predetermined locations. 20. The method according to claim 4, characterized in that the markers contain a polypeptide. 21. The method according to claim 4, characterized in that the markers contain nucleic acid. 22. The method according to claim 4, characterized in that the desorption spectrometry is a laser desorption / ionization mass spectrometry. 23. The method according to claim 20, characterized in that the desorption spectrometry is a laser desorption / ionization mass spectrometry. 24. The method according to item 21, wherein the desorption spectrometry is a laser desorption / ionization mass spectrometry. 25. A kit for detecting analyte in a sample, containing a substrate for desorption spectrometry, containing at least one predetermined location, and each predetermined location contains an adsorbent that resolves the analyte in the selectivity condition provided by the adsorbent and eluant, and an eluant or instructions for processing the sample in condition of selectivity. 26. The kit according to claim 25, wherein the at least one analyte intended for diagnosing the disease is at least one diagnostic marker of the disease. 27. The kit according to p. 26, characterized in that the disease is characterized by many diagnostic markers, and the substrate contains many predetermined locations, and each predetermined location contains an adsorbent that resolves at least one of the diagnostic markers. 28. The kit according to item 27, wherein the at least one adsorbent is a multiplex adsorbent containing such types of adsorbent, each of which delays at least one diagnostic marker. 29. The kit according to item 27, wherein the at least one adsorbent does not contain a biopolymer. 30. The kit according to item 27, wherein the at least one predetermined location contains a ligand specific for the diagnostic marker. 31. The kit of claim 30, wherein the ligand is an antibody. 32. A substrate for desorption spectrometry containing at least one adsorbent in at least one predetermined location, and at least one adsorbent resolves many diagnostic markers of the pathological condition in a sample taken from a patient. 33. The substrate according to p, characterized in that at least one adsorbent does not contain a biopolymer. 34. The substrate according to p, characterized in that one adsorbent detects many diagnostic markers.

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