JP2009288160A - Biosample analysis system, biosample analysis method, biosample pretreatment device, and biosample pretreatment method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosample analysis system and a biosample analysis method, applicable to tandem mass (MS/MS) analysis by LC-MS, having excellent accuracy, and suitable for a biosample, especially a protein, and to provide a biosample pretreatment device and a biosample pretreatment method which are necessary for analysis. <P>SOLUTION: This analysis system comprises the pretreatment device for reacting a plurality of immobilized enzymes acquired by immobilizing decomposition enzymes for a biosample on a support body with the biosample on each different reaction part, and an LC-MS device for performing MS/MS analysis of each reactant acquired by the pretreatment. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

The present invention relates to a biological sample analysis system, a biological sample analysis method, a biological sample pretreatment apparatus, and a biological sample pretreatment method suitable for biological samples, particularly proteins.

Proteins are biosynthesized by transcription and translation in cells and then subjected to various modifications to become functional biomolecules. To date, more than 300 post-translational modifications have been reported, each of which is closely related to the structure, function, localization, interaction, etc. of the protein. Abnormal post-translational modifications that are indispensable for elucidation of internal mechanisms have been reported to be associated with various diseases such as cancer, Alzheimer's disease, diabetes, etc. Post-translational modifications are effective disease markers and drug targets Expected.

  Mass spectrometry (MS) is an effective technique for analyzing post-translational modifications. In protein MS analysis, a protein is usually measured after being decomposed into a peptide by a proteolytic enzyme such as trypsin, and the modification site is identified. For this purpose, it is necessary to select a proteolytic enzyme capable of producing a peptide suitable for MS detection of the modified site. However, constructing a plurality of enzyme reaction systems at the initial stage for the selection is a heavy burden for researchers and the like. For example, there are about 20 candidate enzymes, and general enzyme digestion takes 10 hours for each type. Therefore, it takes 200 hours to examine 20 kinds of enzymes, and it is practically impossible to carry out this examination for each sample.

  In order to overcome this problem, a proteolytic technique using an immobilized enzyme is effective. In this case, by performing the reaction by immobilizing n types of proteolytic enzymes on the same support, it is possible to improve the throughput n times and determine a suitable proteolytic enzyme in a short time. Moreover, since a large number of experiments can be earned in a short time, highly accurate data can be acquired.

As such a method for determining a proteolytic enzyme using a plurality of types of enzymes, (1) JP-A-2006-262829, (2) JP-A-2005-513490 and JP-A-2007-309673 are disclosed.
In (1), a method is reported in which a plurality of types of enzymes are immobilized on a support, and a plurality of types of enzymes and one type of sample are simultaneously reacted inside a single reaction vessel.
In (2), a method of adding a plurality of types of proteolytic enzymes onto a solid-phased MALDI target and analyzing the sample by MALDI-MS is reported.

JP 2005-513490 JP JP 2007-309673 A

However, in the above (1), since the sample is reacted at the same time inside the single reaction tank, the reaction can proceed efficiently, but the problem is that contamination between enzymes occurs and accuracy is lowered.
On the other hand, (2) is an analysis method of MALDI-MS, and the amount of protein required for enzyme reaction is as small as about 0.2 ug, and post-translational modification analysis using a liquid chromatograph mass spectrometer (LC-MS). It is not enough to do.

  The object of the present invention is applicable to tandem mass (MS / MS) analysis by LC-MS, and is a highly accurate biological sample, particularly a biological sample analysis system suitable for proteins, a biological sample analysis method, and a biological sample necessary for the analysis. It is providing the pre-processing apparatus and the biological sample pre-processing method.

In order to achieve the above object, the present invention provides a pretreatment apparatus for reacting a plurality of immobilized enzymes, on which a degrading enzyme of a biological sample is immobilized on a support, with the biological sample in separate reaction units, Each of the reactants obtained from the pretreatment comprises an LC-MS apparatus for MS / MS analysis.

  In order to achieve the above object, the present invention comprises a pretreatment step of reacting a biological sample with a plurality of immobilized enzymes immobilized on a support and the biological sample in a separate reaction part. And an analysis step of performing MS / MS analysis on each of the reactants obtained in the above step using an LC-MS apparatus.

  Furthermore, in order to achieve the above-mentioned object, the present invention provides a pretreatment device for reacting a biological sample with a plurality of immobilized enzymes immobilized on a support and a degrading enzyme of the biological sample. Each of the immobilized enzymes is reacted in a separate reaction section.

  In order to achieve the above object, the present invention provides a pretreatment method for reacting a plurality of immobilized enzymes immobilized on a support with a degrading enzyme of a biological sample with the biological sample. Each of the immobilized enzymes is reacted in a separate reaction section.

  According to the present invention, it is applicable to tandem mass (MS / MS) analysis by LC-MS, and is a highly accurate biological sample analysis system, biological sample analysis method, biological sample pretreatment apparatus and biological sample pretreatment necessary for analysis A method can be provided.

As the best mode for carrying out the present invention, protein analysis will be described as an example. Target proteins include proteins or protein complexes that have been artificially forced and expressed and purified, extracts from cells and tissues or blood (serum, plasma), urine, cerebrospinal fluid, saliva, etc. Extracts from the body and unpurified cells and tissues or blood (serum, plasma), urine, cerebral spinal fluid, saliva, etc. are envisaged.

  Examples of proteolytic enzymes to be immobilized include trypsin, chymotrypsin, endoproteinase ArgC, endoproteinase AspN, endoproteinase GluC, endoproteinase LysC, aminopeptidase, carboxypeptidase, papain, pepsin, plasmin, plasminogen, thrombin, factor X, etc. Is assumed.

  As the shape of the support for immobilizing the proteolytic enzyme described later, a solid flat substrate, beads, and a hollow structure are used, and examples of the material include membranes such as nitrocellulose and PVDF, glass, wafers, and the like. It is possible to use a material such as a resin such as silicon or plastic, a metal, or the like that is subjected to surface modification suitable for fixing as required. Surface modification includes poly-L-lysine and aminosilane that can immobilize the target molecule by physical adsorption, functional groups such as aldehyde group and epoxy group that can immobilize the target molecule by covalent bond, and purpose Avidin or Ni-NTA that can be immobilized using affinity for molecules can be used. In the present invention, an enzyme immobilized on a support is referred to as a solidifying enzyme.

  In the present invention, unlike the conventional case, in order to perform an enzyme reaction on a flat support, a plurality of reaction vessels for reacting a sample on the support are formed. Then, a plurality of proteolytic enzymes are immobilized in different reaction tanks inside the reaction tank and reacted with the sample inside the reaction tank. As reaction vessels, in order to avoid contamination with the sample reaction in the adjacent reaction vessel, it is desirable that each reaction vessel has as few openings as possible during the reaction and is sealed.

  The shape of the reaction vessel is preferably an angleless shape that facilitates convection of the sample inside the reaction vessel and efficiently performs the reaction, for example, a circle or an ellipse. Bubbles generated by convection or the like inside the reaction tank cause a reduction in reaction efficiency. Therefore, in order to suppress this, a shape in which the inner shape of the reaction vessel is partially recessed and the width is narrow is particularly effective. As the required amount of sample increases, the reaction vessel also increases and the number of depressions increases.

  As a material of the reaction vessel, silicone rubber or the like that can be adsorbed on a support is assumed. For example, PDMS (polydimethylsiloxane) is an excellent material with little biological sample adsorption.

  First, a first embodiment will be described with reference to FIGS. FIG. 1 shows an embodiment of a protein analysis system. This protein analysis system includes a pre-enzyme sample storage unit 701 for storing a sample before the enzyme reaction, a sample enzyme reaction unit 702 for enzymatic digestion of the sample, and an enzyme for storing the sample after the enzyme reaction A sample storage unit 703 after the reaction, an LC-MS device 704 for analyzing the sample, a sample introduction unit 705 for introducing the sample from the sample storage unit 701 before the enzyme reaction, and a sample enzyme after the enzyme reaction A sample recovery unit 706 for recovering from the reaction unit 702 to the sample storage unit 703 after the enzyme reaction, and a sample moving unit 707 for introducing the sample from the sample storage unit 703 after the enzyme reaction to the LC-MS device 704 are provided. .

  Here, as the sample introduction unit 705, the sample recovery unit 706, and the sample moving unit 707, a general mechanism including a positioning mechanism and a movable mechanism for moving to the target portion can be used. Further, the pre-reaction sample storage part 701 and the post-reaction sample storage part 703 can also be conventional ones.

  Therefore, the sample enzyme reaction digester 702 and the LC-MS apparatus 704 will be described with reference to FIGS. FIG. 2 shows the steps of protein analysis of this embodiment, and FIG. 3 mainly shows the configuration and process of the sample enzyme reaction digester 702 along the steps shown in FIG. The numbers (1) to (9) in FIGS. 2 and 3 indicate the steps described below.

  This embodiment includes (1) a step of forming an immobilization reaction tank for immobilizing a plurality of types of proteolytic enzymes on a flat support, and (2) immobilization of a plurality of types of proteolytic enzymes on the support. (3) Immobilization of the proteolytic enzyme on the support, (4) Washing the support to remove the non-immobilized proteolytic enzyme, (5) Immobilization A step of forming a sample reaction tank for reacting the proteolytic enzyme with the sample, (6) A step of adding the sample to the inside of the sample reaction vessel, (7) Agitating by applying vibration to the inside of the sample reaction vessel to which the sample has been added And (8) a step of recovering the produced protein degradation product, and (9) a step of performing LC-MS analysis on the recovered protein degradation product. However, the present invention is not limited to the above embodiment.

Hereinafter, each step will be described in detail.
(1) Process of forming an immobilization reaction tank for immobilizing multiple types of proteolytic enzymes on a flat support ProteoChip ^TM (Type A, Proteogen) was used as a support 201 for proteolytic enzyme immobilization . A reaction vessel sheet 202 made of PDMS was attached to the support 201 in advance. The reaction tank sheet 202 is shaped like a mask, and is provided with an elliptical depression that forms a reaction tank at the center of the mask. Therefore, when the reaction vessel sheet 202 is brought into close contact with the support, an immobilized reaction vessel 203 capable of introducing the solution into the space is formed. In order to bring the support 201 and the sheet 202 into close contact with each other, the dedicated holder 205 fixes the both. This is called an immobilization support 220. The capacity of the immobilized reaction tank in this example is 40 μL.

(2) Step of adding plural types of proteolytic enzymes to the inside of the immobilization reaction vessel on the support. Trypsin, endoproteinase LysC (hereinafter LysC), and endoproteinase GluC (hereinafter V8) were used as proteolytic enzymes. The characteristics of each proteolytic enzyme are shown in FIG. Trypsin (T8802, SIGMA) was adjusted to 1 mg / mL using PBS (pH 7.4). LysC (1047825, Roche) and V8 (1047817, Roche) were prepared with sterile water to 20 μg / mL and 10 μg / mL, respectively. 40 μL of three types of proteolytic enzyme solutions 206, 207, and 208 were injected into each reaction tank 203 formed from the solution injection port 204 provided in the reaction tank sheet 202 using a micropipettor 209.

(3) Process of immobilizing the proteolytic enzyme on the support The immobilized support 220 is placed in the sealed container 210, and 200 μL of sterilized water 211 is added to each of the four corners of the sealed container 210 to cover the humidity. In the state kept, it was allowed to stand overnight in a constant temperature bath 212 at 4 ° C.

(4) Step of washing the support to remove the non-immobilized proteolytic enzyme
The support 201 from which the reaction vessel sheet 202 was peeled off from the immobilized support 220 was immersed in a container filled with a cleaning solution 213 (PBS (pH 7.4)), and washed by shaking for 10 minutes with a shaker. After that, it was rinsed twice with 50 mM (M: mol / L) ammonium bicarbonate. Water on the support 201 was removed with a filter paper.

(5) Step of forming a sample reaction vessel for reacting the immobilized proteolytic enzyme with the sample The PDMS reaction vessel sheet 202 of the same shape used in step (1) is pasted on the washed support 201. Then, the reaction support 230 having the sample reaction tank 231 is prepared by fixing with the dedicated holder 205.

(6) Step of adding sample into sample reaction vessel Sample 214 (reduced alkylated BSA or reduced alkylated ovalbumin or β-casein) diluted to 0.1 mg / mL in advance is poured into each sample reaction vessel 231 as a solution. 30 μL was injected from the inlet 204, and a sealed seal was stuck on the inlet 204 to close each sample reaction vessel 231.

(7) Step of performing a proteolytic reaction by stirring by applying vibration to the reaction tank to which the sample has been added In order to efficiently decompose the sample in the sample reaction tank, it is desirable to stir the sample. Therefore, the vibration motor 215 was installed on the upper part of the sample reaction tank 231 and operated, and the sample solution was reacted overnight in the constant temperature bath 212 at 37 ° C. with stirring. As another stirring method, a method of applying ultrasonic waves can be considered.

(8) Process for recovering the produced protein degradation product The vibration motor 215 was stopped, the seal attached to the solution inlet 204 was peeled off, and the sample solution was recovered using the micropipette 209.

(9) Step of LC-MS analysis of recovered protein degradation product The recovered protein degradation product was subjected to MS / MS measurement by LC-MS216 (NanoFrontierLD, Hitachi High-Technologies). Based on the obtained mass data, the protein database NCBIsp was searched by MS / MS ion search using MASCOT (Matrix Science) which is analysis software, and the protein was identified. The conditions for measurement and database search are as follows.

LC section:
Column: Monocap for Fast 50um x 150mmL, tip10um, GL Science
Mobile phase A solution: 2% acetonitrile containing 0.1% formic acid
Mobile phase B liquid: 98% acetonitrile gradient containing 0.1% formic acid: Linear gradient flow rate of liquid A ratio 100% → 60% (liquid B ratio 0% → 40%) 60 minutes after the start of measurement: 0.1 μL / min
MS part:
Ion source: electrospray
Polarity: positive ion mode
Mass scan range: 200-2000
Database: NCBIsp
Taxonomy: Mammalia (for BSA, β-casein), Metazoa (for ovalbumin)
Enzyme: Trypsin or LysC or V8
Missed cleavages: 1
Fixed modifications: Carbamidomethyl (C) (BSA, ovalbumin)
Variable modifications: Oxydation (HMW), Phospho (STY)
Peptide tol: ± 0.3 Da
MS / MS tol: ± 0.1 Da
Instrument: ESI-TRAP

The experimental results obtained in the above steps are shown below.
The protein identification results are shown in FIG. The portion indicated in gray in the sequence corresponds to the peptide detected by MS, and the portion indicated by a box corresponds to the phosphorylated peptide detected. In addition, FIG. 6 shows the number of identified peptides, scores, and sequence coverage of each protein. Different parts of the protein are identified by each enzyme treatment, and the overall coverage is improved by combining three types of data (BSA: 56-76% → 89%, β-casein: 16-21% → 32%, Ovalbumin: 14-51% → 65%). In addition, phosphorylated peptides derived from each protein were confirmed in the LysC degradation product of β-casein and the V8 degradation product of ovalbumin. The MS / MS measurement results of each phosphorylated peptide are shown in FIGS. 7 (A) and 7 (B), respectively.

  From the above results, it is possible to improve the sequence coverage of the protein by combining the analysis results using multiple types of proteolytic enzymes, and the detection of modified peptides such as phosphorylation is the protein used It was found that the use of LysC for the analysis of β-casein phosphorylation and V8 for the analysis of phosphorylation of ovalbumin were optimally affected by the type of degrading enzyme.

  As described above, according to the present embodiment, the multiple enzyme immobilization device can perform the selection of the optimal proteolytic enzyme as described above in a single experiment, so that the protein analysis system with excellent accuracy, An analysis method and a preprocessing device necessary for the analysis can be provided.

  A second embodiment will be described with reference to FIG. FIG. 8 shows an analysis system using a liquid chromatogram column with separate reactions of a plurality of types of enzymes and one type of sample.

  The liquid chromatogram column 503 has a plurality of liquid chromatograms arranged in parallel in the liquid feeding system, and each column is pre-filled with beads, each of which is immobilized with a plurality of types of enzymes, for each type of enzyme. ing. Here, the method of immobilizing the enzyme on the beads may be prepared by putting the beads in the immobilization reaction tank shown in the first embodiment, or by using other conventional methods. Here, using a pump 501, a sample such as protein is introduced into each column via the switching valve 505 from the autosampler 502 and reacted, and after passing through the switching valve 506 again, it is introduced online into the LC-MS 504. To analyze. A plurality of the liquid chromatograms may be directly connected to the liquid feeding system, and a sample may be inserted.

  Also in this embodiment, since a sample can be reacted independently with a plurality of types of enzymes using a column as a reaction tank, a biological sample analysis system, an analysis technique, and a pretreatment device necessary for analysis are provided. can do.

  As a third embodiment, FIG. 9 shows an application example of the present technique in a disease primary screening test for medical examination. In a test using a disease marker protein, a technique for quantifying a specific peptide obtained by treating a marker protein with a proteolytic enzyme is effective. A sample 602 collected from each patient 601 is introduced into the individual reaction tank by the method according to the first embodiment or the second embodiment, and the multiple samples are simultaneously prepared. After decomposing, it is introduced into LC-MS604. Diagnosis 606 of a disease is performed based on the result 605 of quantitative analysis of a peptide having a specific molecular weight from the signal intensity in LC-MS.

  As shown in this embodiment, the present invention can also be applied to a disease primary screening test for health examination.

  The present invention is intended for research in hospitals, pharmaceutical companies, universities, etc. where disease marker search, drug discovery development, protein function analysis is performed, and is required for analysis systems, analysis methods and analyzes suitable for biological samples, particularly proteins Can be provided. In the future, when the disease marker protein is put to practical use in examinations and screenings, it can be provided as a consumable for simultaneously processing a large number of samples with high throughput.

It is a figure which shows embodiment of this invention of a some protein analysis system. It is a figure which shows the process of performing the protein analysis of this embodiment. It is a figure which shows the structure and process of the sample enzyme reaction digestion part 702 mainly along the process shown in FIG. It is a table | surface which shows the characteristic of each proteolytic enzyme. It is a figure which shows the protein identification result by LC-MS. It is a table | surface which shows the protein identification result by LC-MS. It is a figure which shows the phosphorylated peptide MS / MS analysis result by LC-MS. (A) LysC degradation product-derived phosphorylated peptide analysis result of β casein, (B) V8 degradation product-derived phosphorylated peptide analysis result of ovalbumin. It is a figure which shows other embodiment of this invention applied to the online analysis technique by the column for liquid chromatograms which fix | immobilized the proteolytic enzyme. It is a figure which shows other embodiment of this invention applied to the disease primary screening test | inspection in a medical examination.

Explanation of symbols

201: Immobilization support
202: Reaction tank sheet (manufactured by PDMS)
203: Immobilized reaction tank
204: Solution inlet
205: Holder
206: Proteolytic enzyme A
207: Proteolytic enzyme B
208: Proteolytic enzyme C
209: Micro pipettor
210: Sealed container
211: Water drops
212: Thermostatic bath
213: Cleaning solution
214: Sample
215: Vibration motor
216: LC-MS
220: Immobilization support
230: Reaction support
231: Sample reaction tank
501: Pump
502: Autosampler
503: Proteolytic enzyme immobilization column
504: LC-MS
505: Switching valve
601: Health checkup recipient
602: Specimen collected from a health checkup
603: Proteolytic enzyme immobilization device
604: LC-MS
605: LC-MS data
606: Diagnosis result
701: Sample storage before enzyme reaction
702: Sample enzyme reaction section
703: Sample storage after enzyme reaction
704: LC-MS system
705: Sample introduction part
706: Sample collection unit
707: Sample moving part.

Claims (21)

A pretreatment device for reacting a plurality of immobilized enzymes, on which a degrading enzyme of a biological sample is immobilized on a support, with the biological sample in a separate reaction unit, and a reaction product obtained from the pretreatment, respectively, MS / MS A biological sample analysis system comprising an LC-MS device for analysis. 2. The biological sample analysis system according to claim 1, wherein the biological sample is a protein. The pretreatment apparatus has a member that forms the reaction part with the support so that the plurality of immobilized enzymes are provided on the same support, and the immobilized enzymes are individually contained therein. The biological sample analysis system according to claim 2. The biological sample analysis system according to claim 3, wherein the support is a flat substrate, the member is a sheet having a recess, and forms a reaction tank with the recess and the support.   The biological sample analysis system according to claim 2, wherein the reaction unit is a plurality of LC columns, and the immobilized oxygen is separately contained in the LC column.   The biological sample analysis system according to claim 5, wherein the support is a bead.   The biological sample analysis system according to claim 1, wherein a diagnostic material is provided based on the MS / MS analysis result. LC-MS is a pretreatment step in which a biological enzyme degrading enzyme is immobilized on a support and a plurality of immobilized enzymes are reacted with the biological sample in separate reaction units, and the reaction product obtained in the step is LC-MS A biological sample analysis method comprising an analysis step of performing MS / MS analysis by an apparatus. The pretreatment step includes a step in which the plurality of immobilized enzymes are provided on the same support, and a reaction portion forming step in which the support and the reaction portion are formed so that each of the immobilized enzymes is individually present. The biological sample analysis method according to claim 8. The said support body is a plane substrate, The said reaction part formation process has a reaction tank process which has a reaction tank which forms the said reaction part with the sheet | seat which has a recessed part, and the said support body, The said reaction body formation process has a reaction tank process. Biological sample analysis method.   The living body according to claim 11, wherein the immobilized enzyme has a step of forming an immobilization reaction tank formed of a sheet having a recess and the support, and forming the immobilization enzyme in the immobilization reaction tank. Sample analysis method.   The reaction section is a plurality of LC columns, and the reaction section forming step includes a step of forming the immobilized enzymes individually in the LC column, and the pretreatment step is for the previous LC. The biological sample analysis method according to claim 8, further comprising a step of introducing the sample into a column. A pretreatment device for reacting a biological sample with a plurality of immobilized enzymes immobilized on a support and a biological sample, wherein the plurality of immobilized enzymes are reacted in separate reaction sections. A biological sample pretreatment apparatus. The plurality of immobilized enzymes are provided on the same support, and have a member that forms the reaction part with the support so that each of the immobilized enzymes is individually present. The biological sample pretreatment apparatus as described. The biological sample pretreatment device according to claim 14, wherein the support is a flat substrate, the member is a sheet having a recess, and forms a reaction tank with the recess, the support, and the like. 14. The biological sample pretreatment apparatus according to claim 13, wherein each of the different reaction units is provided on the same substrate.   The biological sample pretreatment device according to claim 13, wherein the reaction unit is a plurality of LC columns, and the immobilized oxygen is separately contained in the LC column. A pretreatment method for reacting a biological sample with a plurality of immobilized enzymes immobilized on a support and a biological sample, wherein the plurality of immobilized enzymes are reacted in separate reaction sections. A biological sample pretreatment method. 19. The living body according to claim 18, wherein the plurality of immobilized enzymes are provided on the same support, and the support and the reaction part are formed so that the immobilized enzymes are individually contained therein. Sample pretreatment method. The biological sample pretreatment method according to claim 19, wherein the support is a flat substrate, the member is a sheet having a recess, and forms a reaction tank with the recess and the support. A pretreatment method for reacting a biological sample with a plurality of immobilized enzymes immobilized on a support and a biological sample, comprising a plurality of LC columns, wherein the LC column is provided with the immobilized enzyme A biological sample pretreatment method characterized by comprising oxygenated oxygen separately.

Images