Method for preparing proteomics sample by one-pot method

Abstract

The invention provides a method for preparing a proteomic sample by a one-pot method, which comprises the following steps of: selecting a protein cracking component SDC and a labeled buffer solution HEPES which can be compatibly used, and selecting a corresponding labeled reagent, a protease inhibitor, a reducing agent and an alkylating reagent according to requirements; the steps of protein extraction, enzymolysis, reduction, alkylation, marking and the like can be completed in one pot, and liquid change is not needed during the period, so that the proteomics sample preparation process is simpler and more convenient, the sample preparation time is remarkably shortened, and the efficiency is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of protein sample processing and relates to a method for preparing proteomics samples in a one-pot process. Background Art

[0002] The application of proteomics to the study of clinical samples, including disease prevention, early diagnosis and treatment, is of great significance and has broad development and application prospects. The protein sample preparation of applied proteomics currently mainly includes protein extraction, enzymatic hydrolysis, labeling, IP enrichment and other links. The solutions in different links are often incompatible and need to be carried out under different solvent conditions. There will be various solvent replacements and sample transfer operations during the connection process, and desalting and drying steps are required to obtain clean proteins or peptides, which can easily lead to sample loss and waste of time.

[0003] At present, there are three common mainstream sample pretreatment systems: urea extraction-dilution enzymatic hydrolysis-SPE desalting, detergent lysis solution extraction-precipitation enzymatic hydrolysis-SPE desalting and phenol extraction-dilution enzymatic hydrolysis-SPE desalting. Among them, the phenol extraction method is generally suitable for samples with high plant and lipid content, which requires extraction, precipitation and other steps, which is easy to cause losses and is not suitable for trace sample preparation. The Triton extraction method is generally suitable for clinical samples, but Triton has poor extraction effect on nuclear proteins, and as a detergent, it not only affects the enzymatic hydrolysis effect, but also easily causes serious interference to mass spectrometry analysis if it is not removed cleanly, resulting in poor data quality. Usually, the protein extracted by the Triton extraction method adopts the precipitation enzymatic hydrolysis method, which requires multiple acetone washing steps in the middle, which is easy to cause losses and is not suitable for trace sample preparation. The urea extraction method is generally suitable for cells and animal tissues, and the lysis ability is moderate. When the sample amount is small, the dilution enzymatic hydrolysis method can be used to avoid excessive operations, reduce sample losses, and combined with Zibtip desalting, it can be suitable for trace sample preparation to a certain extent.

[0004] In addition to the above-mentioned sample preparation system, the laboratory of Matthias Mann, an authority in the field of proteomics, also uses another preparation system, the so-called "microreactor", which integrates protein extraction, reduction, alkylation, enzymatic hydrolysis, and desalting, simplifies the intermediate links of sample preparation and reduces sample loss, and is suitable for the preparation of trace samples. The StageTips (STop and GoExtraction Tips) micro-reactor was first proposed by Matthias Mann in 2003. It is used in the sample preparation link in proteomics research. It is simple and easy to operate, and can select suitable fillers according to different needs, and is continuously improved in later applications. However, the reagent materials used in this "microreactor" are relatively complex, and more than 8 different reagents are used successively. The corresponding consumables cost is relatively high, and a single sample treatment costs about 30 euros each time. Based on this technology, PreOmics has developed the iST-NHS kit, which can quickly (~4h) pre-treat multiple samples. However, the iST-NHS kit can only achieve part of the purpose of the present invention. It is still necessary to extract the protein first and then transfer it to the centrifugal column of the kit to achieve solution conversion in different links, and the required operation steps and consumables are relatively more.

[0005] Different from detergents such as Triton and SDS, sodium deoxycholate (SDC) is an ionic detergent that is friendly to enzymatic hydrolysis and mass spectrometry, and has a medium to high protein cleavage ability, and also has a good solubility for membrane proteins. Experimental systems using SDC as a cleavage component have been reported in many papers, and have good omics results. However, there are currently no reports on the use of SDC to truly achieve "one-pot" protein sample processing.

[0006] In summary, based on the shortcomings of the current protein sample preparation system: 1) extraction, enzymatic hydrolysis, labeling and other steps need to be carried out under different solvent conditions, which is not only time-consuming but also easy to cause sample loss; 2) the "microreactor" system uses relatively complex reagent materials, using more than 8 different reagents in succession, and the cost of consumables is relatively high, and a single sample processing costs about 30 euros each time. Therefore, it is urgent to find a protein sample processing method that can truly achieve the "one-pot method" to simplify the operation, improve efficiency and reduce costs. Summary of the invention

[0007] To solve the above problems, the purpose of the present invention is to provide a one-pot method for preparing proteomics samples. By selecting compatible protein lysis components SDC and labeling buffer HEPES, and selecting corresponding labeling reagents, protease inhibitors, reducing agents, and alkylating agents as needed, in the process of proteomics sample preparation, protein extraction, enzymatic hydrolysis, reduction, alkylation, labeling and other steps can be completed in one pot without changing the liquid during the process, making the proteomics sample preparation process simpler, significantly shortening the sample preparation time, and improving efficiency.

[0008] The preparation process of proteomics samples usually includes protein extraction, enzymatic hydrolysis (including precipitation, washing, enzymatic hydrolysis, reduction, alkylation, desalting, and drying), labeling (including peptide dissolution, labeling, desalting, and other steps), IP enrichment, etc. The solution components in different links are often incompatible and require desalting, drying, and other steps to obtain clean proteins or peptides. For example: 1) Protein extraction: The main lysate components in the protein lysate, such as urea, triton, SDS, etc., inhibit enzyme activity and affect enzymatic hydrolysis; buffer salts in the protein lysate, such as Tris and ammonium bicarbonate, will react with the labeling reagent and affect labeling. 2) Enzymatic hydrolysis: Commonly used enzymatic hydrolysis buffers such as ammonium bicarbonate and Tris will react with the labeling reagent and affect labeling; the alkylating agent (iodoacetamide) commonly used during enzymatic hydrolysis needs to be protected from light and has poor compatibility with the entire operating environment.

[0009] Therefore, when using existing methods to prepare proteomics samples, different steps require tube transfer and solvent replacement. Not only is the operation process very cumbersome, it can also cause sample loss or introduce other impurities, leading to sample loss and time waste, and even affecting the accuracy of subsequent test results.

[0010] The present invention provides a system capable of preparing proteomics samples in a "one-pot" manner, unifying the solutions of protein extraction, enzymatic hydrolysis and labeling systems of cells and animal samples, simplifying operations, reducing costs, and thus significantly shortening the sample preparation cycle.

[0011] In one aspect, the present invention provides a method for preparing a protein sample in one pot, the method comprising the following steps:

[0012] (1) adding a protein extract to the sample; the protein extract comprises sodium deoxycholate and a labeling buffer, and the labeling buffer is a HEPES buffer;

[0013] (2) No need to change the solution, just add the enzyme directly for enzymatic hydrolysis;

[0014] (3) No need to change the solution or remove salt, just add the labeling reagent directly for labeling.

[0015] In existing lysis buffers, SDC is often used in combination with other detergents (such as triton, SDS, etc.). Although they are all detergents, triton, SDS, etc. are incompatible with mass spectrometry and generally need to be removed by precipitation enzymatic hydrolysis, FASP enzymatic hydrolysis, etc. The steps are cumbersome and inefficient, and if they are not removed cleanly, it will have a serious impact on subsequent mass spectrometry analysis. In addition, the existing lysis buffer system generally uses a Tris-HCl buffer system, which is incompatible with the subsequent labeling system, and it is impossible to achieve a "one-pot method" for preparing proteomics samples.

[0016] The present invention only uses SDC as the main cleavage component. As a new detergent, SDC can be used for protein extraction and is compatible with enzymatic hydrolysis and mass spectrometry. The present application also adjusts the solution buffer conditions so that the entire system can achieve a unified solution for protein extraction, enzymatic hydrolysis, and labeling, truly realizing the "one-pot method".

[0017] SDC is used as the main lysis component, and HEPES buffer is added to the lysis solution to achieve simultaneous reduction and alkylation during protein extraction, reducing operation time and improving efficiency.

[0018] The present invention combines SDC (sodium deoxycholate) extract with TMT / iTRAQ labeling buffer (HEPES buffer) to design a new mixed buffer ("one-pot" buffer), which can be applied to multiple preparation links such as cell and animal tissue protein extraction, enzymatic hydrolysis, and labeling. Based on the new mixed buffer, the present invention develops a "one-pot" protein sample preparation method flow, which does not require the replacement of the solvent system in the same centrifuge tube, simplifies the operation process, and reduces costs.

[0019] Compared with the classic sample preparation process, the present invention can reduce the sample preparation time from 2 days to 8 hours without affecting the experimental results. Figure 1 As shown, the "one-pot" protein sample preparation method provided by the present invention can save the precipitation protein, protein washing, reduction and alkylation reagents in the enzymatic hydrolysis process, and can also shorten the enzymatic hydrolysis time (from 12 hours to 4 hours to complete the enzymatic hydrolysis). Labeling can be performed without the need for desalting and draining steps, and can save steps such as peptide dissolution in the labeling process.

[0020] By adopting the "one-pot" protein sample preparation method provided by the present invention, the enzymatic hydrolysis time can be shortened from 12 hours to 4 hours, and the reasons are as follows: a. the protein is in a cleavage and extension state under the action of the lysis solution SDC, which is more conducive to the full reaction of the enzyme and the protein; b. a trypsin and lysine endonuclease (Trypsin / Lys-C) double enzyme digestion system is adopted to improve the enzymatic digestion efficiency of lysine in the protein sequence; c. the solution system can be applied to protein extraction, enzymatic hydrolysis, reduction, alkylation, labeling and other links, without the need for desalting, draining and other operations, thus saving operation time.

[0021] In addition, in the "one-pot" protein sample preparation process, since the reduction and alkylation reactions are carried out simultaneously with the enzymatic hydrolysis, that is, the reduction and alkylation reactions are also completed during the enzymatic hydrolysis.

[0022] Since the mixed buffer ("one-pot" buffer) formula of the present invention is suitable for protein extraction, enzymatic hydrolysis, labeling and other links, no additional desalting link is required for solution conversion.

[0023] Compared with the iST-NHS kit of PreOmics, the "one-pot" protein sample preparation method provided by the present invention does not require the use of a centrifuge for solvent replacement operations, the reagent materials used are relatively simplified, and in the same centrifuge tube, there is no need to change the solvent system, which simplifies the operation process and greatly reduces costs.

[0024] Furthermore, the concentration of sodium deoxycholate is 0.2%-2.0%, and the concentration of HEPES buffer is 0.2M.

[0025] In some embodiments, the concentration of sodium deoxycholate is 1.0%.

[0026] Furthermore, the protein extract in step (1) further comprises a protease inhibitor, wherein the protease inhibitor is used to inhibit the degradation of the modified side chain of the protein to be tested.

[0027] In some methods, when the research object is protein acylation modification, inhibitors 3μM TSA and 50mM NAM need to be added; when the research object is protein phosphorylation modification, inhibitor 1% phosphate inhibitor cocktailⅤ needs to be added; when the research object is protein ubiquitination modification, inhibitor 50μM PR-619 needs to be added.

[0028] In some embodiments, the main components of the mixed buffer ("one-pot" buffer) used are: 1% SDC, 0.2M HEPES, and 1% protease inhibitor (depending on the modification research, the corresponding modification inhibitor can be added).

[0029] Furthermore, the protein extract described in step (1) also includes a reducing agent and / or an alkylating agent, wherein the reducing agent is tris-(2-carboxyethyl)phosphine (TCEP, a reducing agent that can break disulfide bonds between / within proteins or polypeptides to form free sulfhydryl groups), and the alkylating agent is chloroacetamide (CAA, an alkylating agent used to block free sulfhydryl groups).

[0030] Furthermore, the pH of the protein extract in step (1) is 8.0.

[0031] Furthermore, the enzyme in step (2) includes trypsin and lysine endonuclease (Lys-C); and the labeling reagent in step (3) is TMT or iTRAQ.

[0032] The present invention adopts the Trypsin / Lys-C double enzyme digestion system, which can significantly improve the enzyme digestion efficiency and shorten the enzyme digestion time.

[0033] The cleavage sites of Trypsin are arginine (R) and lysine (K), but the cleavage efficiency of R is higher than that of K; the cleavage site of Lys-C is K, which can make up for the defect of low cleavage efficiency of Trypsin on K. At the same time, the cleavage conditions of Trypsin and Lys-C are almost the same, so they can be used together to improve the cleavage efficiency and shorten the cleavage time, which has a synergistic effect.

[0034] Of course, the present invention can also use a single enzyme for enzymolysis, but it is necessary to extend the enzymatic cleavage time or increase the number of enzymatic cleavages.

[0035] In some embodiments, the labeling agent in step (3) is TMT and / or iTRAQ.

[0036] On the other hand, the present invention provides a reagent for preparing a protein sample in one pot, comprising sodium deoxycholate and a labeling buffer, wherein the labeling buffer is a HEPES buffer.

[0037] In another aspect, the present invention provides a use of a protein extract for preparing a reagent for one-pot processing of proteomics samples, wherein the protein extract comprises sodium deoxycholate and a labeling buffer, and the labeling buffer is a HEPES buffer.

[0038] In yet another aspect, the present invention provides use of sodium deoxycholate (SDC) for preparing a protein extract for one-pot processing of proteomics samples.

[0039] In another aspect, the present invention provides use of a HEPES buffer for preparing a protein extract for one-pot processing of proteomics samples.

[0040] The one-pot method for preparing proteomics samples provided by the present invention has the following beneficial effects:

[0041] 1) One-pot processing, simple operation, can complete all operations of protein extraction, enzymatic hydrolysis and labeling in one centrifuge tube and one solution system;

[0042] 2) Shorten the sample preparation cycle from 2 days to 8 hours, improve sample preparation efficiency, and increase efficiency by >30%;

[0043] 3) No need to transfer tubes or replace solvents, which can reduce sample loss;

[0044] 4) It is highly versatile and can be used for the preparation of macro and micro cell / animal tissue samples;

[0045] 5) Low cost, no need to introduce additional consumables. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 A schematic diagram comparing the classic sample preparation method and the "one-pot method" preparation process provided by the present invention;

[0047] Figure 2 The electrophoresis diagram of the proteomics samples prepared by the four groups of methods in Example 2;

[0048] Figure 3 A schematic diagram of the protein yield detection results of proteomics samples prepared by the four groups of methods in Example 2;

[0049] Figure 4 It is a schematic diagram of the operation process of the normal system and the one-pot system in Example 3;

[0050] Figure 5 This is the labeling effect test result of the normal system in Example 3;

[0051] Figure 6 This is the labeling effect test result of the one-pot system in Example 3;

[0052] Figure 7 A schematic diagram of the test results of the mass spectrometry repeatability of the sample prepared by the "one-pot method" buffer in Example 7;

[0053] Figure 8 Schematic diagram of the validation results of the "one-pot" buffer preparation for cell samples (left figure, breast cancer cells, biological replicates) and animal tissue samples (right figure, mouse liver, biological replicates) in Example 8. DETAILED DESCRIPTION

[0054] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings. It should be pointed out that the following embodiments are intended to facilitate the understanding of the present invention and do not have any limiting effect on it. All features disclosed in the embodiments of the present invention, or all steps in the methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any way.

[0055] Example 1: One-pot method for preparing proteomics samples provided by the present invention

[0056] 1. Solution preparation

[0057] Protein extract: 1 g of SDC (purchased from Sigma, model 264103), 4.7660 g of HEPES (purchased from Sigma, model H3375), 0.2867 g of reducing agent tris-(2-carboxyethyl)phosphine (purchased from Sigma, model 75259),

[0058] 0.3740g of alkylating agent chloroacetamide (purchased from Sigma, model C0267) was added, and 1ml of protease inhibitor was added (when the research object is protein acylation modification, inhibitors 3μM TSA and 50mM NAM need to be added; when the research object is protein phosphorylation modification, inhibitor 1% phosphate inhibitor cocktailⅤ needs to be added; when the research object is protein ubiquitination modification, inhibitor 50μM PR-619 needs to be added. In this example, only the protein group was studied, and protease inhibitors (purchased from Sigma, model PIC0002) were added, dissolved with 85ml ultrapure water, adjusted to pH 8.0 with sodium hydroxide, and then supplemented with ultrapure water to prepare 100ml of protein extract.

[0059] 2. One-pot method for preparing proteomics samples

[0060] The specific steps for one-pot proteomics sample preparation are as follows:

[0061] 1) Grinding or homogenizing with liquid nitrogen: Take the sample out from -80℃, weigh an appropriate amount of tissue sample (mouse brain tissue or mouse liver tissue) into a mortar pre-cooled with liquid nitrogen, grind it into powder with liquid nitrogen, transfer it to a centrifuge tube and set aside. For samples that do not require grinding and homogenization, protein can be directly extracted.

[0062] 2) Extract protein: add 4-8 times the volume of the sample protein extract, sonicate in ice water for 3 minutes, sonicate for 3 seconds and stop for 5 seconds (sonicate for 3 seconds and stop for 5 seconds, a total of 3 minutes), power 25%, extract protein, 20000g, 4℃ centrifuge for 5 minutes. Take an appropriate volume of sample and determine the protein concentration according to the BCA kit (kit for protein quantification).

[0063] 3) Enzymatic hydrolysis: According to the protein quantification results, 100 μg of protein was taken into a volume of 100 μL, and the insufficient volume was supplemented with the lysis solution in Example 1, and a trypsin / Lys-C protease mixture (purchased from ThermoFisher Scientific, model A41007) was added according to a protein: enzyme = 50: 1 (mass ratio), and enzymatic hydrolysis was carried out at 37° C. for 4 hours.

[0064] 4) Stable isotope labeling: Label the peptides according to the TMT or iTRAQ kit operating instructions. In this example, the TMT kit (purchased from ThermoFisher Scientific, model 90066) is preferably used. The simple operation is as follows: a tube of labeling reagent (0.8 mg) is restored to room temperature and then dissolved in 41 μL of acetonitrile, mixed with the enzymatic solution (protein amount range is 25-100 μg) and incubated at room temperature for 1 hour. The labeled peptides are mixed, desalted, and vacuum freeze-dried for use.

[0065] Example 2: Comparison of protein extraction effects between one-pot method and existing methods

[0066] This example compares the one-pot method for preparing proteomics samples provided in Example 1 with the existing proteomics sample method. The samples used are brain tissue samples and liver tissue samples (taken from mice). The methods are specifically divided into the following three groups:

[0067] 1. Prepare proteomics samples using the one-pot method provided in Example 1;

[0068] 2. Using urea as a lysis reagent:

[0069] 1) Grinding or homogenizing with liquid nitrogen: Take the sample out from -80℃, weigh an appropriate amount of tissue sample (mouse brain tissue or mouse liver tissue) into a mortar pre-cooled with liquid nitrogen, grind it into powder with liquid nitrogen, transfer it to a centrifuge tube and set aside. For samples that do not require grinding and homogenization, protein can be directly extracted.

[0070] 2) Extract protein: add 4-8 times the volume of the sample protein extract, sonicate in ice water for 3 minutes, sonicate for 3 seconds and then stop for 5 seconds, power 25%, extract protein, centrifuge at 20000g, 4℃ for 5 minutes. Take an appropriate volume of sample and determine the protein concentration according to the BCA kit (kit for protein quantification).

[0071] 3) Enzymatic hydrolysis: According to the protein quantification results, take 100 μg of protein, make up the volume to 100 μL with 8 M urea, add dithiothreitol to a final concentration of 5 mM, and reduce at 37 ° C for 60 min. Then add iodoacetamide to a final concentration of 11 mM, and incubate at room temperature in the dark for 45 min. Finally, dilute the urea concentration of the sample to less than 2 M with 0.2 M TEAB. Add pancreatic enzyme at a mass ratio of 1:50 (pancreatic enzyme: protein) and hydrolyze at 37 ° C overnight. Then add pancreatic enzyme at a mass ratio of 1:100 (pancreatic enzyme: protein) and continue to hydrolyze for 4 hours. The peptides hydrolyzed by pancreatic enzymes were desalted with Strata X C18 (Phenomenex) and vacuum freeze-dried.

[0072] 4) Stable isotope labeling: Label the peptides according to the TMT kit instructions. The simple operation is as follows: re-dissolve the peptides with 100 μL 0.5M TEAB; restore a tube of labeling reagent (0.8 mg) to room temperature and dissolve it with 41 μL acetonitrile, mix it with the dissolved peptide solution and incubate it at room temperature for 2 hours, desalt the labeled peptides after mixing, and freeze-dry them in a vacuum.

[0073] 3. Using RIPA as a lysis reagent:

[0074] 1) Grinding or homogenizing with liquid nitrogen: Take the sample out from -80℃, weigh an appropriate amount of tissue sample (mouse brain tissue or mouse liver tissue) into a mortar pre-cooled with liquid nitrogen, grind it into powder with liquid nitrogen, transfer it to a centrifuge tube and set aside. For samples that do not require grinding and homogenization, protein can be directly extracted.

[0075] 2) Extract protein: add 4-8 times the volume of the sample protein extract, sonicate in ice water for 3 minutes, sonicate for 3 seconds and then stop for 5 seconds, power 25%, extract protein, centrifuge at 20000g, 4℃ for 5 minutes. Take an appropriate volume of sample and determine the protein concentration according to the BCA kit (kit for protein quantification).

[0076] 3) Enzymatic hydrolysis: According to the protein quantification results, take 100μg of protein, make up the volume to 100μL with RIPA lysis buffer, add dithiothreitol to make the final concentration of 5mM, and reduce at 37℃ for 60min. Then add iodoacetamide to make the final concentration of 11mM, incubate at room temperature in the dark for 45min. Add 600μL of acetone and vortex to mix, and let stand at -20 degrees for 4h to precipitate protein. 20000g, 4℃ centrifuge for 5min to obtain protein, and the protein precipitate is then dispersed and washed 3 times with 1ml acetone. After each wash, 20000g, 4℃ centrifuge for 5min to obtain protein. The protein precipitate is resuspended with 0.2M TEAB, and pancreatin is added at a mass ratio of 1:50 (pancreatin: protein), and enzymatic hydrolysis is carried out at 37℃ overnight. Then pancreatin is added at a mass ratio of 1:100 (pancreatin: protein), and enzymatic hydrolysis is continued for 4h. The peptides digested by trypsin were desalted with Strata X C18 (Phenomenex) and then freeze-dried in vacuum.

[0077] 4) Stable isotope labeling: Label the peptides according to the TMT kit instructions. The simple operation is as follows: re-dissolve the peptides with 100 μL 0.5M TEAB; restore a tube of labeling reagent (0.8 mg) to room temperature and dissolve it with 41 μL acetonitrile, mix it with the dissolved peptide solution and incubate it at room temperature for 2 hours, desalt the labeled peptides after mixing, and freeze-dry them in a vacuum.

[0078] The proteomics samples prepared by the three methods were subjected to electrophoresis and the protein yield was calculated. The electrophoresis spectra are shown in Figure 2 The protein yield test results are shown in Figure 3 shown.

[0079] according to Figure 2 , a one-pot method was used to prepare proteomics samples, and the types of proteins extracted were basically consistent with those extracted by existing methods.

[0080] according to Figure 3 The one-pot method was used to prepare proteomic samples, and the protein yield was basically consistent with that obtained by the existing method. The protein yield of liver tissue samples was slightly improved compared with the existing method.

[0081] It can be seen that the one-pot method for preparing proteomics samples provided by the present invention can fully achieve the effect of existing methods, and even achieve better results than existing methods.

[0082] Example 3: Labeling efficiency of one-pot system buffer

[0083] In this example, a normal system (according to the method of the second group of Example 2) and a one-pot system (according to the method provided in Example 1) were used to prepare proteomics samples, and TMT kit was used for labeling. The number of proteins, peptides and labeled peptides therein were detected by mass spectrometry, and the labeling efficiency was calculated (labeling efficiency = number of labeled peptides / number of peptides * 100%). The schematic diagram of the operation process is shown in Figure 4 , the test results of the normal system are shown in Figure 5 The test results of the one-pot system are shown in Figure 6 A1 and A2 are HeLa cell samples, and B1 and B2 are HeLa cell samples treated with 10 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 12 hours.

[0084] according to Figure 5 and Figure 6 It can be seen that compared with the existing method, the buffer solution of the one-pot system provided by the present invention has no effect on labeling, and the labeling efficiency is higher, reaching 99.6%.

[0085] It can be seen that the buffer solution of the one-pot system provided by the present invention has no effect on labeling and still has very good labeling efficiency.

[0086] Example 4: Effects of different lysis buffers

[0087] In this example, a one-pot method was used to prepare proteomics samples according to the method provided in Example 1, wherein the lysate used SDC, triton, and SDS as described in Table 1, respectively, with a content of 1%, and compared with urea and RIPA extraction methods (2 and 3 in Example 2), and HeLa cells were used as samples to investigate the effects of different lysate components on the one-pot method for preparing proteomics samples. The number of proteins in the sample before treatment, the number of peptides in the proteomics sample prepared after treatment, and the effective spectrum ratio were detected respectively. The method for detecting the number of proteins and peptides was to use QE Plus 60min gradient mass spectrometry for detection, and the mass spectrometry method was as follows: the peptides were dissolved in liquid chromatography mobile phase A (0.1% (v / v) formic acid aqueous solution) and separated using an EASY-nLC1000 ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient was set as follows: 0-40 min, 9%-26% B; 40-54 min, 26%-35% B; 54-57 min, 35%-80% B; 57-60 min, 80% B, and the flow rate was maintained at 500 nL / min. The peptides were separated by the ultra-high performance liquid phase system and injected into the NSI ion source for ionization and then into the Q Exactive TMThe peptide precursor ions and their secondary fragments were analyzed by mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary mass spectrometry scan range was fixed at 100 m / z, and the secondary scan resolution was set to 35,000. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 10 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 20,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of the precursor ions. The secondary mass spectrometry data were retrieved using Maxquant (v1.5.2.8). Search parameter settings: the database is SwissProt Homo sapiens, a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library is added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cleavage mode is set to Trypsin / P; the number of missed cleavage sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; the maximum number of peptide modifications is set to 5; the mass error tolerance of the primary parent ion of the First search and Main search is set to 20ppm and 5ppm, respectively, and the mass error tolerance of the secondary fragment ion is 0.02Da. Cysteine ​​alkylation is set as a fixed modification, and the variable modification is methionine oxidation and protein N-terminal acetylation. The FDR of protein identification and PSM identification is set to 1%. The detection method of the effective spectrum ratio: the ratio of "MS / MS Identified" to "MS / MS" in the summary document of the search result. The detection results are shown in Table 1.

[0088] Table 1. Comparison of one-pot proteomics sample preparation with urea and RIPA extraction

[0089] Lysis buffer composition SDC (one pot method) Urea RIPA Triton (one pot method) SDS (one pot method) Number of peptides 20607 17126 18810 Not measured Not measured Protein number 3133 2684 2801 Not measured Not measured Effective spectrum ratio 73.76％ 50.18％ 55.67％

[0090] As can be seen from Table 1, to achieve one-pot preparation of proteomics samples, the selection of lysate components is very important. Although SDC, triton, and SDS are all detergents, triton and SDS cannot achieve one-pot preparation because they are incompatible with mass spectrometry and will inhibit enzyme activity and affect enzymatic hydrolysis. Therefore, triton and SDS need to be removed in the subsequent steps, and one-pot preparation cannot be achieved. Urea will also inhibit enzyme activity and affect enzymatic hydrolysis, and it is not compatible with the solution components in the subsequent steps. RIPA, which contains both triton and SDS, is also incompatible with the solution components in the subsequent steps and cannot achieve one-pot treatment.

[0091] In addition, compared with urea and RIPA extraction methods, the one-pot extraction method can obtain more peptides and proteins, and the effect is better.

[0092] Example 5: Effect of SDC concentration

[0093] In this example, a one-pot method was used to prepare proteomics samples according to the method provided in Example 1, wherein SDC was used to prepare protein extracts at 0.2%, 0.5%, 1%, and 2%, respectively, and the results of extraction using urea according to the existing method (according to the method of Group 3 of Example 2) were compared. The sample used was HeLa cell, and the enzyme used was a trypsin / Lys-C protease mixture. The number of proteins in the sample before treatment and the number of peptides in the proteomics sample prepared after treatment were detected respectively. The method for detecting the number of proteins and peptides was to use QE Plus 60min gradient mass spectrometry for detection, and the mass spectrometry conditions were as follows: the peptides were dissolved with liquid chromatography mobile phase A (0.1% (v / v) formic acid aqueous solution) and then separated using an EASY-nLC 1000 ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient was set as follows: 0-40 min, 9%-26% B; 40-54 min, 26%-35% B; 54-57 min, 35%-80% B; 57-60 min, 80% B, and the flow rate was maintained at 500 nL / min. The peptides were separated by the ultra-high performance liquid phase system and injected into the NSI ion source for ionization and then into the Q Exactive TMThe peptide precursor ions and their secondary fragments were analyzed by mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary mass spectrometry scan range was fixed at 100 m / z, and the secondary scan resolution was set to 35,000. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 10 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 20,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of the precursor ions. The secondary mass spectrometry data were retrieved using Maxquant (v1.5.2.8). Search parameter settings: the database is SwissProt Homo sapiens, a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library is added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cutting mode is set to Trypsin / P; the number of missed cutting sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; the maximum number of peptide modifications is set to 5; the mass error tolerance of the primary parent ion of the First search and Main search is set to 20ppm and 5ppm respectively, and the mass error tolerance of the secondary fragment ion is 0.02Da. Cysteine ​​alkylation is set as a fixed modification, and the variable modification is methionine oxidation and protein N-terminal acetylation. The FDR of protein identification and PSM identification is set to 1%. The test results are shown in Table 2.

[0094] Table 2. Effect of different SDC concentrations on enzymatic hydrolysis

[0095] Urea extraction 0.2% SDC 0.5% SDC 1% SDC 2% SDC Number of peptides 16655 20844 20679 20607 18572 Protein number 2673 3130 3149 3133 2907 Effective spectrum ratio 58.88％ 74.02％ 73.02％ 73.76％ 70.49％

[0096] According to Table 2, compared with the existing urea extraction method, the one-pot method for preparing proteomics samples has a higher enzymatic hydrolysis efficiency; by comparing the effects of different SDC concentrations on the enzymatic hydrolysis effect, it can be seen that when the SDC concentration is not more than 1%, it has basically no effect on the enzyme activity, but when the SDC concentration reaches 2%, the enzymatic hydrolysis effect shows a downward trend, so the preferred SDC concentration is 1%.

[0097] Example 6: Effects of different buffers on one-pot proteomics sample preparation

[0098] The choice of buffer system also has a very important influence on whether the one-pot method can be achieved. In this example, the one-pot method is used to prepare proteomics samples according to the method provided in Example 1, wherein the buffers are different buffers as described in Table 3, with a content of 0.2M. HeLa cells are used as samples to investigate the influence of different buffer systems on the one-pot method for preparing proteomics samples. The TMT kit is used for labeling, and the number of proteins, peptides and labeled peptides are detected by QE Plus 60min gradient mass spectrometry, and the labeling efficiency is calculated (labeling efficiency = number of labeled peptides / number of peptides * 100%), and the test results are shown in Table 3.

[0099] Table 3. Effects of different buffers on one-pot preparation of proteomics samples

[0100] Buffer HEPES Tris-HCl Ammonium bicarbonate TEAB Number of peptides 2439 2021 1996 2302 Number of labeled peptides 2425 642 513 2204 Labeling efficiency 99.5％ 31.8％ 25.7％ 95.7％ Protein number 1011 364 283 962

[0101] As can be seen from Table 3, the choice of buffer system is also very important to achieve one-pot preparation of proteomics samples. The existing lysis buffer system generally uses Tris-HCl buffer system, which is incompatible with the subsequent labeling system and cannot achieve the "one-pot method"; ammonium bicarbonate will also react with the labeling reagent, affecting the labeling. The pH of TEAB buffer and HEPES buffer are in the range of 8.0, and are compatible with the solution components of the subsequent links, achieving the unification of solvents for enzymatic hydrolysis and labeling links, but the effect of HEPES is better than TEAB, which may be due to the better compatibility of HEPES with the one-pot system.

[0102] Example 7: Effect of different pH on one-pot preparation of proteomics samples

[0103] In this example, a one-pot method was used to prepare proteomics samples according to the method provided in Example 1, and the pH of the prepared one-pot mixed liquid system was adjusted to investigate the effect of different pH on the one-pot method for preparing proteomics samples. The TMT kit was used for labeling, and the number of proteins, peptides and labeled peptides were detected by QE Plus 60min gradient mass spectrometry, and the labeling efficiency was calculated (labeling efficiency = number of labeled peptides / number of peptides * 100%). The mass spectrometry conditions are as follows: the peptides were dissolved in liquid chromatography mobile phase A (0.1% (v / v) formic acid aqueous solution) and separated using an EASY-nLC 1000 ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient setting: 0-20 min, 9%-26% B; 20-24 min, 26%-35% B; 24-27 min, 35%-80% B; 27-30 min, 80% B, the flow rate was maintained at 500 nL / min.

[0104] After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the NSI ion source for ionization and then analyzed by the QExactiveTM mass spectrometer. The ion source voltage was set to 2.0 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometer scanning range was set to 350-1800 m / z, and the scanning resolution was set to 70,000; the secondary mass spectrometer scanning range was fixed to 100 m / z, and the secondary scanning resolution was set to 35,000. The data acquisition mode used a data-dependent scanning (DDA) program, that is, after the primary scan, the top 10 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometer analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometer, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 20,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometer scanning was set to 30 seconds to avoid repeated scanning of the parent ion. The secondary mass spectrometry data were searched using Maxquant (v1.5.2.8). Search parameter settings: the database was SwissProtHomo sapiens, a reverse library was added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library was added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cleavage mode was set to Trypsin / P; the number of missed cleavage sites was set to 2; the minimum length of the peptide was set to 7 amino acid residues; the maximum number of peptide modifications was set to 5; the primary parent ion mass error tolerance of the First search and Main search was set to 20ppm and 5ppm, respectively, and the mass error tolerance of the secondary fragment ion was 0.02Da. Cysteine ​​alkylation was set as a fixed modification, and the variable modification was methionine oxidation and protein N-terminal acetylation. The quantitative method was set to TMT-6plex, and the FDR of protein identification and PSM identification was set to 1%. The test results are shown in Table 4.

[0105] Table 4. Effect of different pH on one-pot preparation of proteomics samples

[0106] pH 7.0 8.0 9.0 Number of peptides 2266 2304 2212 Number of labeled peptides 1482 2291 1774 Labeling efficiency 65.4％ 99.4％ 80.2％ Protein number 504 932 620

[0107] It can be seen from Table 4 that when preparing proteomics samples by the one-pot method, the pH of the mixed buffer system needs to be maintained in an appropriate range to obtain more efficient detection results. When the pH is 8.0, the number of labeled peptides is the largest and the labeling efficiency is the highest.

[0108] Example 8: Effect of Trypsin / Lys-C double enzyme digestion system on one-pot preparation of proteomics samples

[0109] This example investigates the comparison of the enzymatic digestion effects of the double enzyme digestion system and the single enzyme digestion system. According to the method provided in Example 1, a one-pot method is used to prepare proteomics samples, and Trypsin, Lys-C, and Trypsin / Lys-C are used for digestion, and the digestion time is 4, 12, and 16 hours. Detect the number of proteins, the number of peptides, and the ratio of missed cuts. The detection method of the number of proteins and the number of peptides is to use QE Plus 60min gradient mass spectrometry for detection, and the mass spectrometry method is as follows: The peptides are dissolved in liquid chromatography mobile phase A (0.1% (v / v) formic acid aqueous solution) and separated using EASY-nLC 1000 ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient was set as follows: 0-40 min, 9%-26% B; 40-54 min, 26%-35% B; 54-57 min, 35%-80% B; 57-60 min, 80% B, and the flow rate was maintained at 500 nL / min. The peptides were separated by the ultra-high performance liquid phase system and injected into the NSI ion source for ionization and then into the Q Exactive TMThe peptide precursor ions and their secondary fragments were analyzed by mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary mass spectrometry scan range was fixed at 100 m / z, and the secondary scan resolution was set to 35,000. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 10 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 20,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of the precursor ions. The secondary mass spectrometry data were retrieved using Maxquant (v1.5.2.8). Search parameter settings: the database is SwissProt Homo sapiens, a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library is added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cleavage mode is set to Trypsin / P; the number of missed cleavage sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; the maximum number of peptide modifications is set to 5; the mass error tolerance of the primary parent ion of the First search and Main search is set to 20ppm and 5ppm respectively, and the mass error tolerance of the secondary fragment ion is 0.02Da. Cysteine ​​alkylation is set as a fixed modification, and the variable modification is methionine oxidation and protein N-terminal acetylation. The FDR of protein identification and PSM identification is set to 1%. The calculation method of the missed cleavage ratio: the proportion of the "Missed cleavages" column value of 1 or 2 in the peptides document of the search result, and the detection results are shown in Table 5.

[0110] Table 5. Effects of different enzymes on one-pot preparation of proteomics samples

[0111]

[0112] According to Table 5, compared with the use of Trypsin or Lys-C alone, the dual enzyme digestion system using Trypsin / Lys-C can significantly shorten the digestion time, and the digestion can be completed in only 4 hours, and the number of peptides obtained is also higher. When Trypsin or Lys-C is used alone, it takes 12 hours or 16 hours of enzymatic digestion to obtain a better digestion effect.

[0113] Example 9: Repeatability of sample testing

[0114] In this example, the proteomics samples were prepared in one pot according to the method provided in Example 1. The samples used included A1, A2, B1, B2, C1, C2, D1, and D2, wherein A1 and A2 were HeLa cell samples; B1 and B2 were HeLa cell samples treated with 10 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 12 h; C1 and C2 were normal mouse brain tissue samples; and D1 and D2 were brain tissue samples of mice deprived of sleep for 72 h.

[0115] According to the test results, the intensity, RSD and Pearson between samples were calculated. The results are as follows Figure 7 shown.

[0116] according to Figure 7 It can be seen that the intensity of each sample is around 1 after normalization, indicating that the signal intensity and sample size are consistent; the RSD within the group is less than 10% and the Pearson correlation coefficient is greater than 0.7, indicating that the test results between samples are very repeatable.

[0117] Example 10: Comparison of proteomics sample preparation efficiency between one-pot method and existing methods

[0118] In this example, the one-pot system (according to the method provided in Example 1) and the normal system (according to the method of Group 3 of Example 2) were used to prepare proteomic samples, and actual cell samples (breast cancer cells, biological replicates) and animal tissue samples (mouse liver, biological replicates) were used for verification. The results are shown in Figure 8 As shown, the left figure shows the test results of the quantitative repeatability of breast cancer cell protein (SA is the treatment group, WT is the control group), and the right figure shows the test results of the quantitative repeatability of mouse liver tissue protein (C is the control group, E is the treatment group).

[0119] The number of proteins in the samples before treatment, the number of peptides in the proteomics samples prepared after treatment, and the effective spectrum ratio were detected respectively. The number of proteins and peptides was detected by QE Plus 60min gradient mass spectrometry. The mass spectrometry method is as follows: the peptides were dissolved in liquid chromatography mobile phase A (0.1% (v / v) formic acid aqueous solution) and separated using the EASY-nLC 1000 ultra-high performance liquid phase system. Mobile phase A is an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B is an aqueous solution containing 0.1% formic acid and 90% acetonitrile. Liquid phase gradient settings: 0-40min, 9%-26% B; 40-54min, 26%-35% B; 54-57min, 35%-80% B; 57-60min, 80% B, and the flow rate was maintained at 500nL / min. After the peptides were separated by the ultra-high performance liquid phase system, they were injected into the NSI ion source for ionization and then subjected to Q Exactive TMThe peptide precursor ions and their secondary fragments were analyzed by mass spectrometry. The ion source voltage was set to 2.0 kV, and the peptide precursor ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary mass spectrometry scan range was fixed at 100 m / z, and the secondary scan resolution was set to 35,000. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 10 peptide precursor ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5E4, the signal threshold was set to 20,000 ions / s, the maximum injection time was set to 100 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 30 seconds to avoid repeated scanning of the precursor ions. The secondary mass spectrometry data were retrieved using Maxquant (v1.5.2.8). Search parameter settings: the database is SwissProt Homo sapiens, a reverse library is added to calculate the false positive rate (FDR) caused by random matching, and a common contamination library is added to the database to eliminate the influence of contaminating proteins in the identification results; the enzyme cutting mode is set to Trypsin / P; the number of missed cutting sites is set to 2; the minimum length of the peptide is set to 7 amino acid residues; the maximum number of peptide modifications is set to 5; the mass error tolerance of the primary parent ion of the First search and Main search is set to 20ppm and 5ppm, respectively, and the mass error tolerance of the secondary fragment ion is 0.02Da. Cysteine ​​alkylation is set as a fixed modification, and the variable modification is methionine oxidation and protein N-terminal acetylation. The quantitative method is set to TMT-6plex, and the FDR of protein identification and PSM identification is set to 1%. ; The detection method of the effective spectrum ratio: the ratio of "MS / MS Identified" to "MS / MS" in the summary document of the search result. The detection results are shown in Table 6.

[0120] Table 6. Test results

[0121] Sample Type Preparation method Protein number Number of peptides Effective spectrum ratio Mouse liver One Pot Method 4900 29343 22.9％ Breast cancer cells One Pot Method 5974 38269 26.1％

[0122] According to Table 6, the one-pot method for preparing proteomics samples provided by the present invention, under the premise of obtaining good omics data results, the protein extraction lysis solution and the labeling buffer are unified by adjusting the reagent formula, thereby reducing the reduction, alkylation, desalting and other operations, so that the sample preparation efficiency is improved by >30% ( Figure 1 ).

[0123] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A one-pot method for preparing a protein sample, characterized in that: The following steps are involved: (1) adding a protein extract to the sample; the protein extract comprises sodium deoxycholate and a labeling buffer, and the labeling buffer is a HEPES buffer; (2) No need to change the solution, just add the enzyme directly for enzymatic hydrolysis; (3) No need to change the solution or remove salt, just add the labeling reagent directly for labeling.

2. The method according to claim 1, characterized in that The concentration of the sodium deoxycholate is 0.2%-2%, and the concentration of the HEPES buffer is 0.2M.

3. The method according to claim 3, characterized in that The protein extract in step (1) further comprises a protease inhibitor, wherein the protease inhibitor is used to inhibit the degradation of the modified side chain of the protein to be tested.

4. The method according to claim 4, characterized in that The protein extract described in step (1) further comprises a reducing agent and / or an alkylating agent, wherein the reducing agent is tris-(2-carboxyethyl)phosphine and the alkylating agent is chloroacetamide.

5. The method according to claim 5, characterized in that The pH of the protein extract in step (1) is 8.

0.

6. The method according to claim 1, characterized in that The enzymes in step (2) include trypsin and lysine endonuclease; and the labeling reagent in step (3) is TMT or iTRAQ.

7. A reagent for preparing a protein sample in one pot, characterized in that: The method comprises sodium deoxycholate and a labeling buffer, wherein the labeling buffer is a HEPES buffer.

8. Use of a protein extract for preparing a reagent for one-pot processing of proteomics samples, characterized in that: The protein extract comprises sodium deoxycholate and a labeling buffer, and the labeling buffer is a HEPES buffer.

9. Use of sodium deoxycholate for preparing a protein extract for one-pot processing of proteomics samples.

10. Use of HEPES buffer for preparing protein extract for one-pot processing of proteomics samples.

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