JP3831867B2 - Sample sorting method and apparatus therefor - Google Patents

Description

【００２８】

【００２９】

【００３０】
【発明の効果】
本発明の試料分取方法および装置により、電気泳動法などによって分離された成分試料を同一の基盤上に連続的に分取することが可能となり、分取試料の分析操作が容易になった。また、分取台の位置によって試料成分を識別できるので、多数試料でも取扱いが容易である。更に、顕微鏡下で観察した特定の物質を採取することができる。また、回収液が不要であり、分離試料を液滴として直接分取するので、微量成分であっても確実に分注できる。また、電気泳動力を利用しているので、微量試料の搬送が容易で確実に分注できる。これらは、ごく微量の試料に対応できる。分取された被測定試料に酵素処理、脱塩処理、濃縮処理等の処理が効果的にできる。
さらに、本発明の試料分取方法および装置を他の分析装置と組み合わせることにより、超微量の分離液の分取が非常に簡便且つ確実に可能となり、分離を前提とした超微量での二次分析が安定且つ簡単に達成できる。
【００３１】
【配列表】

【図面の簡単な説明】
【図１】本発明のオンプレート分注法の実施形態を示す図である。
【図２】本発明の分取台の一例を示す図である。（ａ）は正面図、（ｂ）は側面図である。
【図３】泳動成分の検出器出力の一例を示す図である。
【図４】本発明のマイクロピペットによる搬送・分取の一例を示す図である。
【図５】各分画のマススペクトルを示す図である。図中、ａ及びｂは図３のピークａ及びｂに相当する。
【図６】分画ａの消化酵素処理を示す図である。
【図７】分画ｂの消化酵素処理を示す図である。
【符号の説明】
１ 試料の入った容器
２ 正電極
３ キャピラリー（細管）
４ 検出器（ＵＶ検出器）
５ 分取台
６ 負電極
７ 計測・制御装置
８ 位置制御機構[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a sample sorting method and a sample sorting apparatus by electrophoresis, and further relates to an analysis method and apparatus in which this method or apparatus is combined with an analysis process in the next stage.
[0002]
[Prior art]
Capillary electrophoresis (CZE method) is applied by applying an electric field to a solution in a capillary (capillary) having an inner diameter of several tens of microns, or by changing the mobility depending on the size and electrical properties of the sample in the solution. This is a method for separating a mixed sample. Since the CZE method uses very thin capillaries, dispersion of the sample region due to molecular diffusion or the like can be suppressed, and sample components can be separated with very high resolution. Moreover, since the internal volume is very small, it is suitable for trace analysis.
In electrochromatography, the inside of a capillary or the like is filled with a chromatographic resin, and the molecules in the solution sample are driven by an electrophoretic force with a very small amount to interact with the resin surface such as distribution and adsorption. A method for separating components based on the difference in mobility has attracted attention as a micro-separation method different from capillary electrophoresis.
[0003]
Molecules and ionic components separated from the mixture sample by such an electrophoretic force are discharged from the one end of the electrophoresis tube with a time difference for each component when an electrophoresis voltage is applied. When the separated component comes to a specific position in the electrophoresis tube, the time can be known by a detector such as an ultraviolet absorption type. Further, the time of passing through the detector and the time until the component is discharged from one end of the electrophoresis tube (discharge time) are substantially constant for each electrophoresis condition.
Samples separated by the CZE method or electrochromatography are analyzed for each sample to analyze the composition and structure of each separated component. At this time, if a small amount of sample is dispensed into a container, the sample adheres to the container wall, etc. There is a problem that the amount of the sample used for analysis is insufficient.
[0004]
  For this reason, in order to reliably dispense the trace components separated by electrophoresis, a technique for dissolving the separated components in a recovered solution and dispensing them into a test tube is disclosed in, for example, Japanese Patent Laid-Open No. Hei 6-1981.37Is disclosed. However, this method requires a large number of test tubes in the case of analysis of a large number of components, and there is a problem that the identification and handling of the test tubes becomes complicated, and a concentration operation is required before analyzing each sample. There is a problem that it becomes necessary.
  On the other hand, an apparatus for analyzing a component separated by electrophoresis by directly applying it to a mass spectrometer has also been developed (Japanese Patent Laid-Open No. Hei.5-164741). However, in such a case, usually, the outer periphery of the electrophoresis tube is coated with a conductive material, or a metal attachment is attached to the opening, so that the electrophoretic force is exerted to the electrophoresis tube. However, there has been a problem that the electrophoretic components cannot be stably and reliably collected at one point because the coating film is peeled off or the conductivity of the attachment is uneven.
[0005]
On the other hand, the matrix-assisted laser desorption / ionization method (MALDI method) is a method in which a sample compound is mixed with a very light-absorbing molecule called a matrix, and the sample is ionized by laser light. Large energy is given to the molecule, and the matrix and sample compound ions are desorbed. On the other hand, time-of-flight mass spectrometry (TOF / MS method) determines the mass by applying a reverse electric field to the ions and measuring the time for the ions to reach the detector from the electrode surface.
Since this MALDI method can be ionized without imposing a heavy burden on the sample, high-mass substances can be ionized, and the TOF / MS method has no limitation on the mass range of measurement. Deionization-time-of-flight mass spectrometry (hereinafter referred to as “MALDI-TOF / MS method”) is suitable for mass analysis of high mass.
[0006]
In particular, in order to analyze biological materials such as proteins, peptides, DNA, etc., the MALDI-TOF / MS method can quickly and sensitively analyze biological components from low molecular weight to high molecular weight. By combining the MS method with an electrophoresis method such as capillary electrophoresis, it is considered that a highly versatile mass spectrometry method can be performed in which analysis can be performed after separating many kinds of substances. However, there has been a problem in that a small amount of sample cannot be reliably sent to the MALDI-TOF / MS method by the conventional sample separation method by electrophoresis. Furthermore, since the MALDI-TOF / MS method requires a pretreatment operation in which a sample is mixed with a matrix and loaded onto a plate, it is difficult to perform on-line measurement combined with the CE method. As a method of combining a sample separated by electrophoresis with a MALDI-TOF mass spectrometer, there is a method of spraying on a sample plate using an electrospray ionization method. Sometimes not valid.
[0007]
[Problems to be solved by the invention]
The present invention can reliably separate a sample separated or transported by an electrophoretic force, such as an electrophoresis method or electrochromatography, regardless of whether it is a large number of components or a small amount. It is an object to provide a sorting method and apparatus. It is another object of the present invention to provide an analysis method in which components separated by electrophoresis are combined with the next-stage analysis such as the MALDI-TOF / MS method and an apparatus therefor using such a method and apparatus. .
[0008]
[Means for Solving the Problems]
That is, in the present invention, one end of an electrophoresis voltage is applied to a conductive sorting table, and an open end to which an electric field of an electrophoresis carrier such as a glass capillary to which an opposite electric field is applied is not applied (open end of an electrophoresis carrier). ) Are brought into contact with or close to each other, and the solution migrated or scattered by the electric field forms droplets, or a minute volume droplet prepared in advance on the sorting table, between the sorting table and the electrophoresis carrier The above-described problem is solved by separating the electrophoresed solution or component into the liquid droplet so that the open end is conducted by the liquid droplet.
Further, it is possible to reliably perform the next-stage analysis such as the MALDI-TOF / MS method for a very small amount of components separated by the electrophoresis method by such a sorting method.
[0009]
That is, an object of the present invention is to form droplets on a conductive sorting table and bring the open end of the electrophoresis tube into contact with the droplets, thereby accurately opening the open end (more precisely, in the electrophoresis tube). It is an object of the present invention to provide a sample sorting method in which an electrophoretic solution is electrically connected to the sorting table, and the sample is discharged onto the sorting table by components by electrophoretic force. Here, the electrophoretic solution in the electrophoresis tube and the droplet on the sorting table are made to conduct, and in order to ensure conduction by this droplet, metal fibers or glass are transferred from the electrophoresis tube to the sorting table. Conductive or non-conductive auxiliary rods such as fibers may be used. Another object of the present invention is to dispose a detector for detecting a component to be migrated in the electrophoresis tube, and to move the end of the electrophoresis tube or the sorting table by a signal from the detector. It is to provide the above-described sample sorting method in which a desired component is discharged to a desired position of the sorting table. Still another object of the present invention is that the fractionation table also serves as a plate for a MALDI-TOF mass spectrometer, and a sample is discharged on the fractionation table by the above-described sample fractionation method, and the sample is discharged. It is an object of the present invention to provide an analytical method characterized in that a matrix is mixed with components and then subjected to MALDI-TOF mass spectrometry. Still another object of the present invention is to provide a plate for the MALDI-TOF mass spectrometer, wherein a predetermined amount of matrix is placed at a predetermined position on the sorting table, and the sample is placed on the matrix. It is an object of the present invention to provide an analysis method characterized in that a sample is discharged by components by a fractionation method, and the discharged components are mixed with the matrix and subjected to MALDI-TOF mass spectrometry. If necessary, various chemicals (for example, chemicals for enzyme treatment, desalting treatment, concentration treatment, etc.) are applied to the sample discharged by the component by the sample sorting method of the present invention. It may be added or mixed. Such an operation makes it possible to use a very small amount of sample effectively.
[0010]
Another object of the present invention is a sample sorting device comprising an electrophoresis tube and a conductive sorting table, wherein droplets are formed on the sorting table, and the open end of the electrophoresis tube is connected to the open end. A sample fraction in which the open end (to be exact, the electrophoresis solution in the electrophoresis tube) and the sorting table are brought into electrical contact with a droplet, and the sample is discharged onto the sorting table by component by electrophoresis force. It is to provide a take-off device. Still another object of the present invention is to further include a detector for detecting a component that migrates in the electrophoresis tube, and to move the end of the electrophoresis tube or the sorting table by a signal from the detector. It is an object of the present invention to provide a sample sorting apparatus. Another object of the present invention is an analyzer in which the sample fractionator and the MALDI-TOF mass spectrometer are combined, wherein the fractionation table also serves as a plate for the MALDI-TOF mass spectrometer, An object of the present invention is to provide an analyzer characterized in that a sample is discharged onto the plate by the sample sorting device and the discharged component is subjected to MALDI-TOF mass spectrometry. A device for performing various treatments (for example, enzyme treatment, desalting treatment, concentration treatment, etc.) is further added to the sample discharged by the component by the sample sorting device of the present invention on a component basis as necessary. Also good. By adding such a device, it is possible to effectively use a small amount of sample.
[0011]
DETAILED DESCRIPTION OF THE INVENTION
In the sample sorting method of the present invention, droplets are formed on a conductive sorting table, and the open end of the electrophoresis tube is brought into contact with the droplets (precisely, the electrophoresis solution). And the sorting table are conducted, and the sample is discharged on the sorting table by component by electrophoretic force.
The sorting table used in the present invention is conductive and is made of metal, conductive organic or inorganic material, or non-conductive material whose surface is coated or printed with a conductive substance.
The droplets formed on the sorting table are made of an aqueous solution or a conductive liquid, and the droplets are formed by a solution that has migrated or scattered in an electric field, or have previously been manually or automatically placed on the sorting table. Prepare by forming drops.
[0012]
In addition, the electrophoresis tube is made of an organic or inorganic material (for example, fused silica capillary) having extremely low conductivity, and there are a solution, a filter paper, a gel-like substance, an amphoteric carrier and the like as a place to apply an electric field. In the current technology, the electrophoresis of the present invention corresponds to capillary electrophoresis or electrochromatography.
In the present invention, these substances in the electrophoresis tube are exposed at their open ends and come into contact with the droplets on the sorting table, thereby conducting them, and the reference side electrode and the sorting table at the other end of the electrophoresis tube, As a result, an electric field is applied, and the sample in the electrophoresis tube migrates and is separated and reliably discharged to one point on the sorting table for each component.
Samples that can be used in the present invention are molecules that migrate by applying a high piezoelectric field in an aqueous solution or in a polar solvent, such as organic ions having various ions, permanent dipoles or induced dipoles, and organic ions such as surfactants. Organic molecules, proteins, nucleic acids, sugars, or complexes of these that are distributed in aggregates are all electrophoretic solutions that change mobility depending on the molecular structure. It becomes a target. Since the separation of this sample is determined by the difference in mobility of each substance in the electrophoresis tube, the electrophoresis conditions are set according to the purpose of fractionation, and the time for migration to the discharge end is set appropriately according to the difference in substance mobility. And can be sorted.
[0013]
In the present invention, the electrophoresis tube may be provided with a detector to detect the component to be migrated. Although there is no restriction | limiting in particular as this detector, From a viewpoint that it does not affect electrophoresis, an ultraviolet or visible light absorption detector, a quartz light detector, etc. are mentioned. Further, the position where this detector is provided is preferably on the side close to the discharge end of the electrophoresis tube. Since the migration speed of each component is constant depending on the electrophoresis conditions, the time for discharging this component can be accurately predicted after the component to be migrated is detected by this detector. Therefore, the end of the sorting table or the electrophoresis tube may be appropriately moved so that the components separated by electrophoresis are discharged to a desired position on the sorting table. In order to move them by the signal from the detector, ordinary mechanical engineering means may be used.
[0014]
Further, by moving one end of the electrophoresis tube and the sorting table relative to each other in the vertical direction, the open end of the electrophoresis tube and the sorting table can be brought into contact with or separated from each other. Since the sorting table constitutes one of the electrophoresis electrodes, the electrophoresis sample is discharged to the sorting table at the time of contact or liquid contact. When the electrophoresis tube and the sorting table are separated from each other, no migration voltage is applied, and thus the sample in the migration tube stops migration and discharge. When the open end of the electrophoresis tube is relatively moved in the horizontal direction while being separated from the sorting table, droplets are formed at different locations on the sorting table, and component samples can be dispensed. Thereby, since a multi-component sample can be dispensed on one sort stand, handling becomes easy and reliable.
[0015]
In this way, the sample separation method or apparatus of the present invention makes it possible to reliably discharge components separated by electrophoresis to one point on the sorting table for each component. Various operations can be performed, and various continuous analyzes can be performed by combining with various analyzers.
Since the separation component is spotted on the plate, the method of the present invention cleaves proteins, nucleic acids, sugar chains, etc. by digestive enzymes after separation (in this method, including complete separation as well as rough separation). Alternatively, selective molecular capture after rough molecular separation using the primary structure analysis of molecules by fragmentation, antigen-antibody reactions such as protein chips, or selective binding of biomolecules, and desorption of the captured molecules by matrix addition Structural analysis, nucleic acid typing by complementary binding on a gene or nucleic acid chip after rough molecular separation, and structural analysis analysis of these molecules after desorption by addition of matrix (At this time, digestive enzymes may be used in combination) Yes, it can be done on the plate as it is.
[0016]
Examples of the analysis apparatus and method that can be combined with the sample sorting method or apparatus of the present invention include, for example, a protein sequencer, DNA sequencer, biological component separation / identification system using MALDI-TOF mass spectrometry, or conductivity. Examples thereof include a gene chip, a protein chip, an affinity chip and the like configured on a base material (for example, a diamond thin film).
Among these, the method of the present invention is particularly suitable for a sample stage that must be conductive.
In particular, when the MALDI-TOF / MS method is combined with capillary electrophoresis, components separated by electrophoresis are discharged to a sorting table that also serves as a plate for a MALDI-TOF mass spectrometer. When a predetermined amount of the matrix is previously spotted or spray-dried at a predetermined position of the substrate or droplets of the matrix solution are formed, the components and the matrix can be easily mixed.
[0017]
The laser normally used for MALDI-TOF / MS is N₂Laser (337 nm) and Er-YAG laser (2940 nm), but Nd: YAG (1064 nm), dye (tunable in the visible region), YAG-OPO (630 nm), CO₂Lasers such as (10600 nm), alexandrite (755 nm), Ar (488 to 514 nm), holmium-YAG (20100 nm) can also be used.
As the matrix, an organic acid or an organic base that absorbs light from an excitation light source (mainly a pulse laser) can be used. For example, N₂The laser (337 nm) includes CHCA (α-cyano-4-hydroxycinnamic acid), DHBA (2,5-dihydroxybenzoic acid), 3-HPA (3-hydroxypicolinic acid), HABA (2- (4-hydroxyphenylazo) -benzoic. acid), sinapinic acid (3,5-dimethoxy-4-hydroxy-cinnamic acid), organic acids such as picolinic acid, HARMAN (2-methyl-β-carboline; 1-methyl-9H-pyrido [3 , 4-b] indole), HARMINE (2-methyl-β-carboline; 1-methyl-9-acethyl-pyrido [3,4-b] indole), HARMOL (2-methyl-β-carboline; 1-methyl -9-hydroxy-pyrido [3,4-b] indole) and other β-carbolines, THAP (2,4,6-trihydroxyacetophenone monohydrate), ATT (6-aza-2-thiothymine) and other purines and pyrimidines ( Organic base), cobalt ultrafine particles (trade name Co-UFP), porous silica and the like can be used. For the Er-YAG laser (2940 nm), glycerol, ice, succinic acid, or the like can be used.
[0018]
The amount of the matrix is appropriately determined according to the sample and the purpose, but in many cases, it is used as an organic solvent solution near the saturation concentration of the matrix in order to promote crystallization.
After the components thus mixed with the matrix on the plate are naturally dried or dried in a desiccator or the like, the plate is mounted in a MALDI-TOF mass spectrometer. Thus, by combining the MALDI-TOF / MS method with capillary electrophoresis, high-speed molecular analysis that has not been conceived in the past, such as simultaneous analysis of molecular identification by pre-separation and molecular primary structure analysis, becomes possible.
[0019]
Hereinafter, the present invention will be specifically illustrated, but is not intended to limit the present invention.
An example of the sample sorting device of the present invention is shown in FIG.
In capillary electrophoresis, a sample of a mixture and a solvent (electrophoretic solution) are sequentially introduced into a capillary 3 from a container 1 in which the sample is placed. The inner diameter of this capillary is usually about 30 to 200 μm. As the electrophoresis solution, a volatile salt, for example, 0.01 M ammonia / ammonium formate buffer 1 microliter is usually used. A voltage is applied between the positive and negative electrodes 2 and 6. This voltage is usually about 1 to 20 kV. When the mixture is electrophoresed in the capillary 3, the components in the sample are separated, and the detector 4 detects that each component sample has passed. Thereafter, the component sample is discharged from one end of the capillary 3 after a certain time.
The position control mechanism 8 has a function of controlling the position of the plate 5 having a large number of sample mounting portions on the upper surface in the XY biaxial directions. The position control mechanism 7 only needs to be able to position the plate so that an arbitrary sample mounting portion is within the operating range with reproducibility of about 0.1 mm in the XY directions. Since the time interval of intermittently appearing components may be short, it is preferable to have a servo mechanism capable of positioning at high speed. The position control mechanism 7 further has a function of raising and lowering the height of the plate. What is necessary is just to be able to position the relationship between the narrow tube and the plate at two positions of contact and separation.
[0020]
The measurement / control device 7 supplies an electrophoretic voltage between the positive and negative electrodes. Further, the output of the detector 4 is converted to measure the presence / absence of separation components and the passage time. Further, the position control mechanism 8 is instructed to change the position / height of the plate.
An example of the plate 5 is shown in FIG. This plate is made of a conductive material (for example, gold-plated stainless steel, stainless steel, diamond thin film, etc.). In addition, an electrophoretic voltage wiring can be connected to the negative electrode 6. In this example, there is a sample mounting portion composed of 5 × 5 = 25 indentations, and continuous analysis of 25 components of the mixture is possible at one time. If the sample to be collected is a small amount, a flat plate may be used even if there is no dent.
[0021]
An example of the sorting method of the present invention will be described with reference to the detector output (FIG. 3) and FIG. In the sorting method of the present invention, a conductive liquid (which may be an electrophoretic solution) is first placed in the form of droplets on the sorting table (A). When the conductive sorting table constituting one of the electrophoresis electrodes is moved (or one end of the capillary tube may be moved), and the liquid on the sorting table is brought into contact with one end of the capillary tube, the electrophoresis A voltage is applied and migration begins. Until the detector detects the component, the liquid in part (A) is only the electrophoresis solution, and is removed by suction with a device (not shown). First, the necessary fractionation component (a) is detected and moved relatively before the discharge time elapses to cancel the liquid contact state between (A) and the thin tube. At this time, the electrophoresis stops. When one end of the sorting table or capillary tube is moved and another part (B) on the sorting table is brought into contact with one end of the capillary tube, the first required sorting component (a) is liquid. Sorted into drops (B). Next, when the necessary component (b) is detected, the liquid contact state is released before the time of discharge similarly passes, and then it is brought into contact with another portion (C). In this way, by repeating the relative movement, a plurality of components are sorted at different positions on the same sorting table.
[0022]
When the MALDI-TOF / MS method is used to analyze the composition and structure of each separation component, it is preferable that the MALDI plate has a shape that can be shared with the plate of the present invention. It is also possible to place a MALDI matrix in advance on the plate at the dispensing position. This is advantageous because the electrophoretic sample is dispensed onto the matrix, allowing mass spectrometry to be performed continuously.
The sample collected according to the present invention can be chemically processed for each sorting position. Therefore, different processing can be performed on the plate for each separated component, which is suitable for automation.
[0023]
  The capillary electrophoresis described above is shown as an example of a component separation method. If the components can be separated by electrophoresis and discharged with a time difference, the method and apparatus of the present invention can be used in electrochromatography. The present invention can also be applied to other separation methods and apparatuses. Furthermore, the sample sorting method of the present invention can be used not only for an apparatus for separating components, but also for transporting trace components when an electrophoretic force can be applied. For example, as shown in FIG. 4, a minute sample sucked by a micropipette or the like is held in a thin tube, and the thin tube is once immersed in a solvent container provided with a reference electrode, and the sorting table of the present invention is used at the other end. For example, the suction sample can be dispensed on the sorting table for each suction sample. In the example of this figure, the tip of the capillary is doubled, but it is possible to collect samples continuously in any number of stages, and the tip of the capillary uses the bend of the entire glass capillary, or Teflon at the root(Registered trademark)The position of the tip can be moved by connecting a tube to create a moving part. In this way, many samples can be collected continuously in a short time compared to the conventional pipette collection method in which the pipette is sucked and discharged from the same mouth in the forward and reverse directions each time.
[0024]
【Example】
Example 1
In order to separate and dispense two kinds of proteins by electrophoresis, the measurement system shown in FIG. 1 was used. In a container 1 having a capacity of 1.5 cc, two kinds of proteins, 1.7 mg of myoglobin (horse, Fw: 17.2 kDa) and 1.4 mg of lysozyme (chicken protein, Fw: 14.3 kDa) were electrophoresed (20 mM pyridine- NH₃A solution prepared by mixing and dissolving in 1 ml of a solution (pH 8.0) was prepared. One end of a capillary 3 (made of fused silica, inner diameter 50 μm) having a total length of about 60 cm is immersed in the container 1. A MALDI plate having an electrode at one end as shown in FIG. One drop of the electrophoresis solution was dropped with a dropper at a desired position on the sorting table 5, and the other end of the capillary 3 was placed in contact with the droplet. Further, a UV detector 4 (E-800 manufactured by JASCO Corporation, measurement wavelength: 280 nm) was disposed in the middle of the capillary 1 so that proteins migrated from the outside of the capillary 1 could be detected. The length of the capillary from the sample introduction end to the detector 4 was about 40 cm, and the length from the UV detector 4 to the sorting table 5 was about 20 cm. The positive electrode 2 was inserted into the container 1, the negative electrode 6 was taken from the sorting table 5, and an electric field of 167 V / cm was applied between the two electrodes. FIG. 3 shows the absorption by the UV detector after applying the electric field. In FIG. 3, a is an absorption peak due to lysozyme and b is myoglobin. From the time from the start of electrophoresis to the time when these proteins are detected by the UV detector 4, the time when the sample is discharged to the sorting table 5 is predicted, and the signal from the UV detector 4 is measured and controlled 7. In this way, the dispensing position on the sorting table 5 was changed by the position control mechanism 8 every 30 seconds, and 10 sections of separated samples were prepared continuously for 5 minutes until the detection of the protein was completed.
[0025]
Example 2
The 10-section separated sample obtained in Example 1 was air-dried, and 1 μL of α-cyano-4-hydroxycyanic acid (10 mg / mL) solution was added dropwise as a matrix to the sample elution part. This sample was measured as a MALDI-TOF / MS using a Voyager RP manufactured by PEBiosystem. As shown in FIG. 3, two peaks a and b are separated by CZE, and the results of mass analysis of the sample corresponding to these peaks are shown in FIG. 5. From each mass analysis, the first peak is lysozyme (m / Z 14,311), the second peak could be identified as the eluted myoglobin (m / z 17,237).
[0026]
Example 3
Further, the components a and b separated in the same manner as in Example 1 were each subjected to digestive enzyme treatment, and sequence analysis was performed from the mass spectrum. The latter MALDI-TOF / MS was performed in the same manner as in Example 2. Samples (myoglobin (a), lysozyme (b)) eluted on the plate were digested with an enzyme by adding trypsin and chymotrypsin, respectively, and then an α-cyano-4-hydroxycyanic acid (10 mg / mL) solution as a matrix Was added, and the sequence was performed from the mass spectrum data. The amino acid sequences of myoglobin and lysozyme could be determined from the results of mass spectrometry shown in FIGS.
Conventionally, in order to determine the amino acid sequence of each protein contained in a sample in which several kinds of proteins are mixed, it has been necessary to perform mass spectrometry through a separate process of purifying each protein. By using it, it becomes possible to perform protein separation and mass spectrometry in one step without going through such conventional complicated steps, and for that purpose, such analysis with a very small amount of sample can be performed reliably and easily. It became possible to do.
[0027]
Hereinafter, the results of the mass spectrometry shown in FIGS. 6 and 7 and their amino acid sequences are shown. In addition,

[0028]

[0029]

[0030]
【The invention's effect】
According to the sample sorting method and apparatus of the present invention, it is possible to continuously sort component samples separated by electrophoresis or the like on the same substrate, and the analysis operation of the collected sample becomes easy. Further, since the sample components can be identified by the position of the sorting table, even a large number of samples can be handled easily. Furthermore, a specific substance observed under a microscope can be collected. In addition, since no recovery liquid is required and the separated sample is directly collected as droplets, even a trace amount component can be reliably dispensed. In addition, since the electrophoretic force is used, a very small amount of sample can be easily and reliably dispensed. These can cope with a very small amount of sample. Treatments such as enzyme treatment, desalting treatment, and concentration treatment can be effectively performed on the collected sample to be measured.
Furthermore, by combining the sample sorting method and apparatus of the present invention with other analyzers, it is possible to very easily and reliably sort a very small amount of separation liquid. Analysis can be achieved stably and easily.
[0031]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a diagram showing an embodiment of an on-plate dispensing method of the present invention.
FIG. 2 is a diagram showing an example of a sorting table according to the present invention. (A) is a front view, (b) is a side view.
FIG. 3 is a diagram illustrating an example of a detector output of an electrophoretic component.
FIG. 4 is a diagram showing an example of conveyance / sorting by the micropipette of the present invention.
FIG. 5 is a diagram showing a mass spectrum of each fraction. In the figure, a and b correspond to peaks a and b in FIG.
FIG. 6 is a diagram showing digestive enzyme treatment of fraction a.
FIG. 7 shows digestive enzyme treatment of fraction b.
[Explanation of symbols]
1 Container with sample
2 Positive electrode
3 Capillary
4 Detector (UV detector)
5 Preparatory stand
6 Negative electrode
7 Measurement and control equipment
8 Position control mechanism

Claims (7)

A droplet is formed on a conductive sorting table, and the electrophoretic solution in the electrophoresis tube is brought into conduction with the sorting table by bringing the open end of the electrophoresis tube into contact with the droplet. A sample sorting method in which components are discharged on the sorting table by electrophoresis force. A detector for detecting the component to be migrated is disposed in the electrophoresis tube, and the end of the electrophoresis tube or the sorting table is moved by a signal from the detector, so that a desired component is transferred to the sorting table. The sample sorting method according to claim 1, wherein the sample is discharged to a desired position. The fractionation table also serves as a plate for a MALDI-TOF mass spectrometer, a sample is discharged onto the fractionation table by the method according to claim 1 or 2, and a matrix is mixed with the discharged component. An analysis method characterized by being subjected to post-MALDI-TOF mass spectrometry. The sorting table also serves as a plate for a MALDI-TOF mass spectrometer, a predetermined amount of matrix is placed at a predetermined position on the sorting table, and a sample is placed on the matrix by the method according to claim 1 or 2. An analysis method characterized in that the components are discharged separately and the discharged components are mixed with the matrix and subjected to MALDI-TOF mass spectrometry. A sample sorting device comprising an electrophoresis tube and a conductive sorting table, wherein droplets are formed on the sorting table, and the electrophoretic tube is brought into contact with the droplets by bringing the open end into contact with the droplets. A sample sorting device for conducting an electrophoretic solution in an electrophoresis tube and the sorting table and discharging the sample by component onto the sorting table by electrophoresis force. 6. The sample fractionation device according to claim 5, further comprising a detector for detecting a component that migrates in the electrophoresis tube, and capable of moving the end of the electrophoresis tube or the sorting table by a signal from the detector. Taking device. An analyzer combining the sample sorting apparatus according to claim 5 and a MALDI-TOF mass spectrometer, wherein the sorting table also serves as a plate for the MALDI-TOF mass spectrometer. An analyzer, wherein a sample is discharged onto the plate by a collecting device according to components, and the discharged components are subjected to MALDI-TOF mass spectrometry.

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