JP2019105608A - Protein-containing sample pretreatment method for protein analysis using mass spectrometer - Google Patents

Abstract

To provide a biological sample pretreatment method which enables highly sensitive and highly accurate protein analysis using a mass spectrometer.SOLUTION: A protein-containing sample pretreatment method for protein analysis using a mass spectrometer, includes a first step (i) in which a protein-containing sample is subjected to reductive alkylation and thereafter a protein-containing fraction is purified, a second step (ii) in which the protein-containing fraction obtained in the first step is subjected to ultrasonic treatment in buffer solution which substantially does not contain protein denaturation agents, and a third step (iii) in which the ultrasonic-treated matter obtained in the second step is subjected to decomposition treatment using a protein digestion enzyme.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method of pretreatment of a protein-containing sample for protein analysis using a mass spectrometer.

  In recent research in the fields of medicine and life sciences, there is a demand for a technique for quantitatively analyzing proteins comprehensively, with high sensitivity and high accuracy. In particular, mass spectrometers can analyze multiple components simultaneously, and in recent years they have greatly advanced in both sensitivity and accuracy, and are used routinely in these research fields (Patent Document 1). However, in quantitative analysis of proteins using a mass spectrometer, it is not uncommon for the pretreatment process to greatly affect detection sensitivity and quantitative reproducibility. In addition, in large-scale protein quantitative measurement with a mass spectrometer, it is necessary to pay attention to the stability of the analysis sample after pretreatment because the analysis time is long. Regarding the pretreatment method, various methods and improvements have been reported. For example, in Patent Document 1, after TCA precipitation of a cell extract, protein modification treatment is performed in Tris buffer containing guanidine hydrochloride, and then reductive alkylation treatment is performed to inactivate the alkylating agent. Thereafter, the protein was digested (decomposed) with trypsin. The resulting digested product is desalted with a column, concentrated by centrifugation, redissolved in iTRAQ buffer, labeled with stable isotope, and used in LC-MS / MS analysis. Alternatively, cell lysates are purified by TCA / acetone precipitation, then sonicated in a buffer without protein denaturing agent, followed by protein digestion with trypsin for 16 hours at 37 ° C., and then After reductive alkylation treatment and subsequent inactivation treatment of the alkylating agent, protein digestion treatment is again carried out at 37 ° C. for 3 hours to completely digest the protein, and the resulting digest is desalted with a column And methods used for nano-HPLC / MS / MS analysis have been reported (Non-patent Document 1). However, there are still many problems in order to carry out large-scale quantitative protein measurement at high sensitivity and high accuracy practically.

Patent No. 5468073

Kim et al. Journal of Proteome Research 2006, 5, 3446-3452 Matsumoto M. et al. Nature Methods 2017, 3, 251-258

For example, in the pretreatment method disclosed in Non-Patent Document 2, it is shown that the protein is denatured by a protein denaturant, purified by methanol / chloroform extraction, and then the denatured protein is digested with protein digesting enzyme and reductively alkylated. However, the process for inactivating the alkylating agent is essential, and it was considered that the problem was that the protein was not digested enough to simultaneously analyze many kinds of trace proteins. . In addition, even with the pretreatment method disclosed in Non-Patent Document 1, reductive alkylation is performed between protein digestion steps with two protein digestion enzymes, and the enzyme treatment time is not long under completely denaturing conditions. It was considered that there is a problem in that the step of inactivating the alkylating agent is essential as in Non-Patent Document 2 as well.
Therefore, it is an object of the present invention to provide a pretreatment method which enables highly sensitive and accurate protein analysis using a mass spectrometer, in particular, a pretreatment method for measuring a trace amount of protein in a biological sample. It will be an issue.

  As a result of intensive studies to solve the above problems, the present inventors conducted the following steps (i) to (iii) in large-scale protein quantitative analysis using a mass spectrometer to detect trace amounts of proteins in biological samples. As a result, the present invention has been accomplished by finding that pre-treatment can be measured with high sensitivity and high accuracy.

That is, the present invention can be exemplified as follows.
[1] A method for pretreatment of a protein-containing sample for protein analysis using a mass spectrometer,
(I) a first step of subjecting the protein-containing sample to reductive alkylation and thereafter purifying the protein-containing fraction,
(Ii) a second step of sonicating the protein-containing fraction obtained in the first step in a buffer substantially free of a protein denaturant,
(Iii) A third step of degrading the sonicate obtained in the second step with a protein digestion enzyme,
Method including.
[2] The method according to [1], wherein the sample is a biological sample.
[3] The method according to any one of [1] to [2], wherein the second to third steps are performed in the presence of an adsorption inhibitor.
[4] The method according to any one of [1] to [3], wherein the protein analysis using the mass spectrometer is a large scale protein quantitative analysis.
[5] The method according to any one of [1] to [4], wherein two or more proteins are simultaneously measured in protein analysis using the mass spectrometer.

  According to the present invention, for example, the protein in the sample solution is reductively alkylated, purified, and then the protein is dispersed and enzymatically digested by sonication, so the alkylating agent is not inactivated. It has become possible. Therefore, by simplifying the pretreatment method, it is possible to achieve loss of protein loss and suppression of working time, working error and reagent cost due to the small number of working steps. In addition, by performing sonication and enzyme digestion in a buffer substantially free of protein denaturant, protein indigestion is improved and a high detection rate can be achieved. Furthermore, the enzyme processing time is shortened, and not only sensitization and precision improvement of protein analysis using a mass spectrometer, but also shortening of the time required for the whole pretreatment method, facilitation of work and suppression of cost, samples ( Control of protein degradation) can be achieved. Therefore, according to the present invention, improvement in detection sensitivity and improvement in quantitative reproducibility are achieved by reducing loss of protein in the sample and improving digestion efficiency in the pretreatment step, and it is also necessary to perform the pretreatment step. By shortening the time, the working days can be shortened and the stability of the sample can be improved. Therefore, a mass spectrometer can be used to analyze proteins easily and with high sensitivity and high accuracy. In particular, high-sensitivity and high-accuracy analysis of a trace amount of protein in a biological sample is possible, so that large-scale protein analysis, particularly large-scale protein quantitative analysis, is highly effective.

The graph which shows the result of having compared the ionic strength of each digested peptide at the time of measuring culture cell origin protein digested material by DDA (Data-dependent acquisition) method. Vertical axis: pretreated sample according to the present invention (Example 2), horizontal axis: sample subjected to enzyme digestion in diluted denaturant solution (without ultrasonication) and then reductively alkylated (comparative example). The figure which shows the chromatogram at the time of measuring Pyruvate kinase muscle (PKM) in a cultured cell by MRM (Multiple Reaction Monitoring) method. Left) Pretreated sample according to the present invention (Example 2), right) Sample subjected to enzyme digestion in diluted denaturant solution (without sonication) and then reductively alkylated (comparative example). The results of LC-MS / MS measurement immediately after preparation are shown for samples prepared under each condition when PEG20000 is added as an adsorption inhibitor (left: PEG) and when PEG20000 is not added (right: water). Figure. After storage at 10 ° C. for 4 days, LC-MS / MS measurement was performed again to verify the decrease in the detected protein peak intensity. (Relative Intensity = calculated as peak intensity after day / peak intensity immediately after preparation).

Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to these embodiments.
In the present invention, analysis and measurement are synonymous unless otherwise noted, meaning both qualitative and quantitative.

  The method of the present invention is a method for pretreatment of a protein-containing sample in protein analysis using a mass spectrometer. The method of the present invention comprises the steps of (i) subjecting a protein-containing sample to reductive alkylation and then purifying the protein-containing fraction, and (ii) the protein-containing fraction obtained in the first step. A second step of sonicating in a buffer solution substantially free of denaturing agent, (iii) a third step of degrading the sonicated product obtained in the second step with a protein digestion enzyme Process.

  The "protein-containing sample" that can be used in the present invention is not particularly limited as long as it is a sample containing a protein. For example, it may be a sample containing a protein of biological origin or a sample containing an artificially synthesized protein. Moreover, the "biological sample" which can be used by this invention means the sample containing the component derived from a biological body. Examples of biological samples include whole blood, plasma, serum, urine, feces, saliva, sputum, semen, tears, nasal discharge; vaginal, nasal, rectal, pharyngeal and urethral swabs, excrement and secretion, and cells , Tissues (eg, biopsy tissue samples) and the like. It may be obtained directly from a living organism or may be prepared in vitro. The living body is not particularly limited as long as it is a living body having a protein, and examples thereof include humans, monkeys, mice, rats, rabbits, hamsters, goats, pigs, dogs, cats, ferrets, cattle, sheep, and horses, etc. .

  The protein which can be measured in the present invention is not particularly limited. The term "protein" also includes embodiments called peptides, oligopeptides or polypeptides. The molecular weight of the protein has no upper limit in size as long as it can be digested with protein digestion enzyme, and the lower limit may be any size that can be used for analysis, for example, preferably 100 Da or more, more preferably 500 Da or more It may be. The protein may be a protein derived from a living body or a protein derived from in vitro. Examples of the in vitro-derived protein include protein components contained in a medicine administered to a living body, and metabolites thereof. The protein may exhibit any localization mode, for example, a protein derived from a membrane, a protein derived from a cytoplasm, or a protein derived from extracellular. Proteins may be measured alone, or two or more may be measured simultaneously. According to the present invention, since a minute amount of protein can be measured with high sensitivity and high accuracy, not only one type of protein can be measured with high sensitivity and high accuracy, but also a plurality of proteins can be measured simultaneously. For example, biological samples containing many types of proteins can be used for large scale protein analysis. Furthermore, it is particularly preferable because it can perform not only identification but also quantification with high sensitivity and high accuracy.

  The pretreatment method of the present invention will be described below by taking the case of cells as a sample as an example, but this is an example of available modes and is not interpreted in a limited manner.

(I) Reductive Alkylation Treatment and Purification Step As the reductive alkylation step in the pretreatment method of the present invention, the cell extract may be subjected to reductive alkylation treatment and then purification of the protein-containing fraction may be mentioned.
Cell extracts can be prepared according to known methods that can be used in general protein analysis methods. For example, those obtained by solubilizing cells with a lysis solution can be mentioned. As the lysis solution, for example, it is possible to use a buffer containing a chaotropic reagent typified by urea or guanidine hydrochloride, a surfactant such as SDS, CHAPS, Triton X-100 or the like, Tris-HCl or the like. For example, the lysate can be added to a cultured cell pellet, and agitation and sonication can be performed to solubilize components in the cell to obtain a cell extract containing the target protein component.

The reductive alkylation treatment means performing a reductive treatment and an alkylation treatment, preferably performing an alkylation treatment after the reductive treatment, but can be performed according to a known method. The reducing agent used for the reduction treatment is not particularly limited as long as it is used for reduction of a protein-containing sample, but TCEP (Tris 2-carboxyethyl) phosphine
and DTT (Dithiothreitol). The alkylating agent used for the alkylation treatment is not particularly limited as long as it is used for alkylation of a protein-containing sample, and examples thereof include 2-iodoacetamide and the like.
For example, a reducing agent such as TCEP or DTT is added so as to give a final concentration of 5 mmol / L, and the reduction alkylation treatment is carried out at 30 ° C. for several hours at 37 ° C. An alkylating agent such as iodoacetamide is added for 30 minutes to perform the alkylation treatment.

  Purification of the protein-containing fraction after reductive alkylation can be performed according to a known method. Examples thereof include acetone precipitation, methanol / chloroform extraction, TCA precipitation, and a combination method thereof, or a solid phase extraction method represented by a purification column. In particular, since the precipitation purification method can precipitate many proteins nonspecifically, it can easily remove alkylating agents that adversely affect protein digestion efficiency using proteins or protein analysis using a mass spectrometer. preferable. In addition, since proteins are taken out as precipitates immediately after alkylation, reagents and time required for the alkylating agent inactivation treatment can be omitted, which is preferable. Furthermore, in the pretreatment method of the present invention, the protein in the cell extract is first subjected to a reductive alkylation step to give a complete denaturing condition in which the disulfide bond of the protein is cleaved, thereby maximizing the digestion efficiency in the subsequent enzyme digestion Because it can be There is no need to prepurify the protein prior to the reductive alkylation treatment, and at least one purification of the protein may be performed, thereby reducing the loss of protein that can be used for the protein analysis (high recovery of protein) Since the amount of work is small, errors in work can be reduced, and the reproducibility of the analysis can be improved, which is preferable.

(Ii) Ultrasonication Step The ultrasonication step in the pretreatment method of the present invention includes the protein-containing fraction purified in the reductive alkylation step in a buffer solution substantially free of a protein denaturant. Ultrasonication may be mentioned. Substantially free of protein denaturing agent means that no protein denaturing agent is included or, if it is included, less than the concentration required for protein denaturation.
The conditions for the ultrasonic treatment may be any conditions that allow the precipitation of the protein to be dispersed, and can be appropriately set by those skilled in the art from known information, for example, under cold temperature (for example, 4 to 15 ° C.)
And using an ultrasonic crusher (for example, Bioruptor (manufactured by Diagenode)). The conditions of the ultrasonic crusher include, for example, ultrasonication at 300 W or more for 5 to 600 seconds, preferably 10 to 400 seconds, more preferably 30 to 300 seconds, and also for 5 to 30 seconds. It is also possible to intermittently repeat a plurality of times with the sound wave and the interval pause of 5 to 30 seconds as a set. Intermittent multiple repetition is preferable because ultrasonic treatment can be performed while confirming the dispersion state of the precipitate of the protein in a state in which excessive damage to the protein is unlikely to occur.

  As the buffer that can be used, the type and concentration thereof can be appropriately selected from known buffers and used, for example, the buffer that can be used in the next enzyme treatment step is preferable. For example, 50 mmol / L ammonium bicarbonate pH 8, 50 mmol / L Tris pH 8, 50 mmol / L triethylammonium bicarbonate (TEAB) pH 8.5 etc. may be mentioned. Use of these is preferable because a buffer that can be used for later analysis of proteins using mass spectrometry can be selected. Since the dispersive effect of the protein is high and the protein denaturing agent is substantially not contained, by maximizing the efficiency of enzyme digestion in the next decomposition treatment step, protein analysis using a mass spectrometer can be performed simultaneously. It is preferable because many types of proteins can be analyzed with high sensitivity and high accuracy.

  In addition, to the buffer solution, for example, an adsorption inhibitor such as polyethylene glycol (PEG), dimethyl sulfoxide (DMSO), Acetonitrile (ACN) Anionic Acid Labile Surfactant I (AALSI) can be added. Since adsorption of protein to a container can be suppressed, even a sample stored for a long time can be measured with high sensitivity, which is preferable. Those skilled in the art can appropriately select and use known substances that inhibit the adsorption of a protein to a container.

(Iii) Decomposition treatment step In the decomposition treatment step in the pretreatment method of the present invention, the sample treated in the ultrasonic treatment step is converted into a peptide fragment that can be analyzed by a mass spectrometer by decomposition treatment with a protein digestion enzyme. . The decomposition treatment can be performed according to a known method. For example, enzymes to be used include trypsin, chymotrypsin, Lys-C, Asp-N, Glu-C and the like, preferably trypsin and Lys-C. The temperature conditions for the degradation incubation are 15 ° C. to 90 ° C., preferably 20 ° C. to 50 ° C., more preferably 37 ° C., and the incubation time is 30 minutes or more, preferably 2 hours. The above, more preferably 3 hours can be mentioned.

  The degraded sample can be used as it is or for protein analysis using a mass spectrometer in addition to known protein purification such as a desalting column. Also, depending on the protein analysis method used, protein labeling can be performed. For example, iTRAQ and mTRAQ (manufactured by AB Sciex) described in Patent Document 1 and TMT (manufactured by Thermo) may be mentioned. Since it is possible to use a buffer solution corresponding to various protein analysis continuously in a series of operations of the ultrasonic treatment step and the decomposition treatment step, a step for the purpose of buffer substitution, protein purification, etc. You can lose It is preferable because the loss of the sample can be suppressed and the working process can be simplified and the working efficiency is high.

The sample subjected to the pretreatment method of the present invention can be used for protein analysis using a mass spectrometer (MS). It can also be used for protein analysis combining a liquid chromatograph (LC) device and a mass spectrometer (MS). The liquid chromatograph and the mass spectrometer may be connected in series with each other or may be independent devices. For example, the components separated by liquid chromatography can be subsequently massed by using an LC-MS system, which can be used an LC-MS system, configured by connecting a liquid chromatograph and a mass spectrometer in series. It can be analyzed. Those skilled in the art can appropriately select, design and use from protein analysis using a known mass spectrometer.

  Examples of protein analysis using a mass spectrometer that can be used in the present invention include protein identification by peptide mass fingerprinting, quantitative analysis of individual or multiple molecules by MRM (Multiple Reaction Monitoring), and shotgun analysis. Typical examples include data-dependent acquisition (DDA) analysis, and data-independent acquisition (DIA) analysis represented by SWATH. In particular, MRM, DDA, DIA and the like are preferable because it becomes possible to simultaneously measure various types of proteins contained in a sample with high sensitivity and high accuracy. In general, MRM, DDA, DIA, etc. are also known as large-scale protein analysis and large-scale protein quantitative analysis.

  As a mass spectrometer, a known mass spectrometer can be used, but in particular, one that can be connected in series to the LC device is preferable because it can be conveniently used. The number of mass spectrometers used may be one, or two or more. Two or more mass spectrometers can be used in series. That is, the LC-MS system may be, for example, LC-MS / MS or LC-MS / MS / MS, with two or more mass spectrometers connected in series. Specifically as LC-MS / MS, QTRAP6500 (made by AB Sciex) is mentioned, for example.

  As a detection method in the mass spectrometer, for example, an ion trap type, a quadrupole type, a quadrupole tandem type, an ion trap quadrupole hybrid type, a sector type, a time of flight type, and a quadrupole time of flight hybrid type It can be mentioned. Examples of ionization methods in mass spectrometers include electrospray (ESI), atmospheric pressure chemical ionization (APCI), matrix-assisted laser desorption / ionization (MALDI), fast atom bombardment (FAB), photoionization (APPI) And sound velocity ionization (SSI). The detection method and the ionization method can be appropriately selected according to various conditions such as the type of protein to be measured.

  Tandem quadrupole mass spectrometers (MS / MS, MS / MS / MS, etc.) include a quadrupole (Q1) functioning as a mass filter, a quadrupole (Q2) functioning as a collision (collision) cell, and This is a mass spectrometer in which quadrupoles (Q3) functioning as mass filters are arranged in series. The tandem quadrupole mass spectrometer can also optimize the ion transmission to obtain the sensitivity required for diagnostic testing. That is, Q1 and Q3 function as a mass filter (mass separator) by applying high frequency voltage and direct current voltage, and by passing voltage and frequency, only ions in a specific mass range can be passed. it can.

  Specifically, for example, in the quadrupole (Q1), from a plurality of ions generated by ionization, based on the mass-to-charge ratio (m / z) of the ions, a specific parent ion capable of reaching the required sensitivity Select (Precursor ion). Next, a selected parent ion (precursor ion) is collided with an inert gas such as argon in the collision cell (Q2) to generate a daughter ion (product ion) (collision induced dissociation: CID). Then, daughter ions (product ions) having a specific mass-to-charge ratio (m / z) are selectively detected by the quadrupole (Q3). The structural information of the object of analysis can be derived from the difference in mass between the detected product ion and the precursor ion.

Therefore, when using a tandem quadrupole mass spectrometer, by repeatedly selecting the precursor ion and the product ion, the protein to be measured is separated while the impurities contained in the biological sample and the protein to be measured are separated. It can be measured with high sensitivity.

  Also, when using a mass spectrometer such as MS / MS / MS, measurement is performed by selectively detecting secondary cleavage ions (second product ions) generated by further cleavage of the selected product ions. The target substance can be further narrowed down to improve the measurement sensitivity.

  Since the detection ratio (ion ratio) of the ions obtained by mass spectrometry is a value unique to the substance, the ion ratio obtained by the analysis of the standard product is compared with the ion ratio obtained by the analysis of the biological sample to obtain the biological sample. The proteins contained in can be identified.

  In addition, in Q1 and / or Q3, by selectively passing two or more ions in separate channels, two or more proteins contained in the biological sample can be analyzed together. Specifically, for example, by selectively detecting two or more product ions generated in the collision cell (Q2) in another channel in Q3, the same mass-to-charge ratio (m Two or more proteins that produce a precursor ion of / z) can be analyzed together.

  The protein to be measured can be quantified based on the result of mass spectrometry. The quantification of protein can be performed by a conventional method. Specifically, for example, the protein to be measured can be quantified based on the peak area ratio obtained by dividing the peak area value of the detected protein to be measured by the peak area value of the internal standard substance whose concentration is known.

  The LC apparatus, mass spectrometer, and various elements included in them can be appropriately selected according to various conditions such as the type of protein to be measured and the type of contaminants with reference to the analysis conditions exemplified above.

  EXAMPLES The present invention will next be described in more detail by way of examples, which should only be taken as an aid for obtaining specific recognition of the present invention, thereby limiting the scope of the present invention in any way. It is not a thing.

Example 1: Large-scale protein measurement procedure by the pretreatment method of the present invention The following examples were carried out according to the following procedure, unless otherwise specified.
Add 200 μl of lysate (7 M Urea, 2% SDS, 100 mM Tris-HCl pH 8.8) to 1.2 × 10 ⁶ cultured cell frozen pellets and stir.
2. Ultrasonic treatment (10 ° C., 10 minutes) to solubilize solid components.
3. Add 200 μL cold water and stir.
4. Treat with ultrasound (10 ° C., 10 minutes).
5. Centrifuge (4 ° C. 15000 G for 5 minutes) to remove insolubles.
6. Crude protein concentration is determined by BCA assay.
7. Aliquot 100 μg of crude protein.
8. After addition of 5 μL of reducing agent (200 mM TCEP), incubation is carried out at 37 ° C. for 30 minutes.
9. After addition of 5 μL of an alkylating agent (400 mM 2-Iodoacetamide), incubation is carried out at room temperature for 30 minutes in the dark.
10. Add 1000 μL of ice cold acetone and stir.
11. Let stand at -30 ° C for 30 minutes.
12. Centrifuge (4 ° C 15000G for 5 minutes).
13. Remove the supernatant and wash the precipitate with ice cold 90% acetone.
14. Centrifuge (4 ° C 15000G for 5 minutes).
15. Remove the supernatant and wash the precipitate with ice cold 90% acetone.
16. Centrifuge (4 ° C 15000G for 5 minutes).
17. The supernatant is removed and 100 μL of 50 mM triethylammonium bicarbonate, 0.001% polyethylene glycol 20000 solution is added.
18. Process ultrasound (set of 10 ° C., 30 sec ultrasound and 30 sec interval rest 5 times).
19.1 μL of LysC or Trypsin solution (1 mg / mL) is added and incubated for 3 hours at 37 ° C.
Add 20.1 μL Trypsin solution (1 mg / mL) and incubate at 37 ° C. for 3 hours.
21. Measure digested peptide concentration in BCA assay.
22. Each protein quantitative value is calculated by LC-MS / MS measurement.

  This pretreatment method eliminates the need for an inactivating agent for the alkylating reagent and eliminates the need for purification or desalting treatment for removing these reagents, which is advantageous in time and cost, and improves data reproducibility as well. It has become possible. In addition, by digesting the protein after reductive alkylation in an adsorption inhibitor-added environment without a denaturing agent, it has become possible to improve the digestion efficiency and to improve the protein detection rate and sensitivity.

Example 2: Improvement of digestion efficiency Example 2 and the comparative example are represented by DDA or DIA analysis by LC-MS / MS, or mTRAQ label between step 21 and step 22 in the method of example 1. Isotopic labeling was performed and MRM analysis was performed.

  In general, in enzyme digestion of proteins, after protein purification, they are redissolved in a solution containing protein denaturants such as urea, guanidine hydrochloride and surfactants, etc., and the activity of digestive enzymes is not reduced by diluting them. It often digests under conditions. However, it has been found that, in fact, the remaining denaturing agent affects the activity of digestive enzymes to a large extent even when the solution is diluted, resulting in dyspepsia. In addition, during enzyme digestion, it was thought that digestion under the fully denatured conditions after cleavage of protein disulfide bonds and alkylation was desirable.

  Using HeLa cells, prepared protein digests were DDA and MRM analyzed according to the method of Example 1, and after reductive alkylation, the effect of digestion efficiency under protein denaturant-free conditioning was verified.

  As a comparative example, the method of Non Patent Literature 2 was carried out, that is, 8 and 9 of Example 1 were performed after 20, and re-dissolved and enzyme-digested with 875 mM guanidine hydrochloride and 62.5 mM triethylammonium bicarbonate solution instead of 17. . When guanidine hydrochloride is added, the protein is solubilized, so the ultrasonic process 18 is not performed.

  The results are shown in FIG. In comparison with the case where each pretreatment product was measured three times each in DDA analysis, the detection intensity of the peptide containing enzyme-misdigested (missed cleaved) was reduced by applying the present invention, resulting in complete digestion. The detection intensity of the peptide increased. The results show that the pretreatment of the present invention can improve dyspepsia.

  In the comparison by MRM method, the target protein could not be detected in the denaturing agent-digested sample (right), but as shown in FIG. 2, a clear peak derived from PKM was observed by the pretreatment of the present invention, and the protein identification And it became possible to quantify.

  In both the DDA method and the MRM method, it is considered that digestion efficiency was improved by performing enzyme digestion in a buffer containing no denaturing agent, to a protein which was first reductively alkylated and completely denatured.

Example 3: Addition of adsorption inhibitor Generally, in protein analysis using mass spectrometry such as LC-MS / MS, measurement takes longer than analysis using an antigen-antibody reaction such as ordinary ELISA. There are many. Therefore, adsorption to the wall of the vial containing the sample may occur between sample pretreatment and analysis, which may affect the analysis result. It is also assumed that adsorption to resin surfaces such as microtubes will occur during pretreatment. Here, according to Example 1, the adsorption suppressing effect was verified by adding PEG 20000 as an adsorption inhibitor from the ultrasonic treatment step. Therefore, what pre-processed on the conditions which do not add PEG20000 was prepared. Both samples were subjected to LC-MS / MS measurement immediately after preparation, stored at 10 ° C. for 4 days, and then measured again to examine the amount of change in protein detection intensity immediately after preparation.

  The detected proteins were classified according to retention time (LC retention time), and in terms of molecular hydrophobicity and adsorption loss rate, as shown in FIG. 3, it is particularly hydrophobic in the pretreatment sample without PEG 20000 (water) Although significant loss occurred in the high molecular weight group (Retention Time 25 min or later), the treatment with PEG 20000 addition had almost no effect. Therefore, by adding PEG 20000 from the pretreatment process, it has become possible to reduce the adsorption loss of the protein and to improve detection sensitivity and quantitative reproducibility.

  ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to measure a large-scale protein quantitative measurement practically, and can be used for the drug development etc. based on the exhaustive analysis of the protein relevant to a disease, and its analysis.

Claims (5)

A method for pretreatment of a protein-containing sample for protein analysis using a mass spectrometer, comprising:
(I) a first step of subjecting the protein-containing sample to reductive alkylation and thereafter purifying the protein-containing fraction,
(Ii) a second step of sonicating the protein-containing fraction obtained in the first step in a buffer substantially free of a protein denaturant,
(Iii) A third step of degrading the sonicate obtained in the second step with a protein digestion enzyme,
Method including.   The method according to claim 1, wherein the sample is a biological sample.   The method according to any one of claims 1 to 2, wherein the second to third steps are performed in the presence of an adsorption inhibitor.   The method according to any one of claims 1 to 3, wherein the protein analysis using the mass spectrometer is a large scale protein quantitative analysis.   The method according to any one of claims 1 to 4, wherein two or more proteins are simultaneously measured in protein analysis using the mass spectrometer.