A method for pre-treatment of plasma protein by mass spectrometry

Abstract

Most of the drugs entering the peripheral circulation will bind to the proteins in the blood, which is a key factor that restricts the primers from entering the target organs and cells to play a therapeutic function. Therefore, comprehensive detection of proteins in peripheral blood plasma is an important measure to study the metabolic process of drugs in vivo and their efficacy, and the comprehensive detection of plasma proteins is mainly through mass spectrometry analysis, but the technology is interfered by the high abundance in plasma, which has always been a problem in the field at home and abroad. The present invention discloses a mass spectrometry pretreatment method for protein in plasma. The present invention effectively precipitates high-density proteins such as globulins in plasma by polyethylene glycol 6000, and then separates the albumin components in plasma by using Cibazin blue gel column, and then performs chromatographic fractionation by reverse liquid chromatography on each of these separated components, and finally uses liquid chromatography-mass spectrometry to perform mass spectrometry detection of proteins for mass spectrometry detection. Through this method, the number of protein detections can be effectively increased by 38.3%, greatly improving the detection depth, and providing a more comprehensive laboratory basis for drug metabolism and efficacy research.

Description

Technical Field

[0001] The invention belongs to the technical field of plasma protein spectrum analysis and relates to a mass spectrometry detection pre-treatment method for proteins in plasma. Background Art

[0002] The detection of molecular markers in peripheral blood is an important basis for disease diagnosis, efficacy observation and prognosis judgment. For example, nucleic acid detection of pathogens, tumor markers such as alpha-fetoprotein and interleukin-6, and inflammatory factors are the main laboratory diagnostic clues for clinical diseases such as infection, tumors, and inflammation. With the development of medicine, these traditional indicators can no longer meet the requirements for accurate diagnosis of diseases, nor are they conducive to the discovery of new diseases. Therefore, the discovery of new molecular markers is an important task in clinical laboratory research.

[0003] The use of mass spectrometry technology to conduct comprehensive detection of proteins in serum or plasma (proteomic analysis) is one of the important methods for discovering protein markers of diseases in peripheral blood, and is also an important research direction in the international field. However, there is a common technical bottleneck of insufficient detection depth, resulting in insufficient number of detected proteins. This is because the content of protein markers related to clinical diseases, such as endocrine hormones, tumor markers, cytokines, inflammatory mediators, etc., in peripheral blood is very low, generally in the range of ng to pg / mL, while the levels of albumin, globulin, coagulation factors, complement, etc. in serum / plasma are very high, generally around mg / mL, almost a million times and more than a billion times that of common disease markers. These high-abundance proteins will greatly reduce the chances of low-abundance proteins being detected, seriously interfering with the detection of these disease-related proteins. Therefore, improving the depth of mass spectrometry detection of serum / plasma to increase the chances of low-abundance proteins being detected is an important issue faced by the international field. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a mass spectrometry pre-treatment method and a treatment kit for protein in plasma in view of the deficiencies of the prior art.

[0005] Purpose of the invention: The present invention aims to increase the number of protein detections in human plasma, mainly by separating high-abundance proteins in plasma samples through polyethylene glycol and Cibacron Blue, so as to greatly increase the possibility of discovering clinical disease markers. Polyethylene glycol 6000 (PEG6000) is added to the plasma sample, and some high-abundance proteins such as globulin, lipoprotein, complement, etc. in the plasma are precipitated. The plasma supernatant after precipitation is filtered using a Cibacron Blue (Cibacron Blue 3G-A) gel column, and the albumin in the plasma supernatant sample will be adsorbed on the gel column. After these two steps, the main high-abundance proteins in the plasma sample are mostly removed, and then the precipitation, the filtrate after removing albumin, the plasma and other samples are chromatographed and fractionated by reverse phase liquid chromatography (RPLC) under alkaline pH conditions, and finally the mass spectrometry protein is detected by liquid chromatography-mass spectrometry, thereby increasing the number of protein detections in the plasma sample.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] The present invention discloses a method for pre-processing mass spectrometry detection of proteins in plasma, comprising the following steps:

[0008] (1) Polyethylene glycol precipitated plasma and albumin removal: the plasma sample is mixed with a surfactant, and then polyethylene glycol precipitated plasma is added to obtain a first mixed solution; the first mixed solution is centrifuged to obtain a precipitate A and a supernatant A; a portion of the supernatant A is centrifuged through an albumin removal centrifugal column to remove albumin, and a supernatant filtrate A is obtained; another portion of the supernatant A is taken and polyethylene glycol precipitated plasma is added to obtain a second mixed solution; the second mixed solution is centrifuged to obtain a precipitate B and a supernatant B; the supernatant B is centrifuged through an albumin removal centrifugal column to remove albumin, and a supernatant filtrate B is obtained;

[0009] (2) Sample precipitation: the plasma sample is mixed with anhydrous acetonitrile for precipitation, and the precipitate C is collected; trichloroacetic acid is added to the supernatant filtrate A and the supernatant filtrate B for precipitation, respectively, and the supernatant filtrate A and the supernatant filtrate B are centrifuged respectively, and the precipitates are washed respectively, and then centrifuged again to obtain precipitates D and E respectively;

[0010] (3) proteolysis and reduction alkylation: the precipitate B, the precipitate C, the precipitate D, and the precipitate E are subjected to proteolysis treatment, protein reduction treatment, protein alkylation treatment, and quenching reaction treatment in sequence to obtain peptide samples;

[0011] (4) Peptide desalting: the peptide sample obtained in step (3) is loaded onto a desalting column, and a desalting washing solution is used for desalting washing, and then a desalting eluent is added for elution, and the eluent containing the peptide is collected and dried to obtain the desalted peptide;

[0012] (5) Reverse liquid phase chromatography fractionation under alkaline conditions: the desalted peptides obtained in step (4) are subjected to reverse liquid phase chromatography fractionation, and the peptide fractions obtained are collected and dried;

[0013] (6) Mass spectrometry detection and protein identification: The peptide samples collected by fractionation in step (5) are injected into a liquid chromatography-mass spectrometer for mass spectrometry detection; the raw mass spectrometry data are analyzed using a protein database.

[0014] The operation steps of the mass spectrometry pretreatment method for plasma protein detection can be found in Fig.10 .

[0015] In some embodiments, in step (1), the albumin removal centrifugal column is a Cibacin blue agarose gel column; the plasma is human plasma or animal plasma; the surfactant is 5% to 10% Triton X-100; the volume ratio of the plasma sample to the surfactant is 9:1; the polyethylene glycol exists in the form of a solution, the solute in the polyethylene glycol solution is PEG6000, and the solvent is a PBS buffer with a pH of 7.3 to 7.5; the mixed solution obtained by mixing the plasma sample and the surfactant is mixed with a polyethylene glycol solution with a PEG6000 concentration of 18 to 22% g / mL to obtain a first mixed solution containing a PEG6000 concentration of 8 to 12% g / mL; the supernatant A described in another part is continuously added with a polyethylene glycol solution with a PEG6000 concentration of 30% g / mL to precipitate the plasma to obtain a second mixed solution containing a PEG6000 concentration of 20% g / mL.

[0016] In some embodiments, preferably, in step (1), the albumin removal centrifugal column is a Cibacin blue agarose gel column; the plasma is human plasma or animal plasma; the surfactant is 10% Triton X-100; the volume ratio of the plasma sample to the surfactant is 9:1; the polyethylene glycol exists in the form of a solution, the solute in the polyethylene glycol solution is PEG6000, and the solvent is a PBS buffer with a pH of 7.4; the mixed solution obtained by mixing the plasma sample and the surfactant is mixed with a polyethylene glycol solution with a PEG6000 concentration of 20% g / mL to obtain a first mixed solution containing a PEG6000 concentration of 10% g / mL; the supernatant A described in another part is continuously added with a polyethylene glycol solution with a PEG6000 concentration of 30% g / mL to precipitate the plasma to obtain a second mixed solution containing a PEG6000 concentration of 20% g / mL.

[0017] In some embodiments, further preferably, in step (1), the albumin removal centrifugal column is a Cibacron Blue 3G-A agarose gel column;

[0018] Wherein, in step (1), after the first mixed solution is mixed at room temperature for 10 minutes, it is centrifuged at 20,000g for 5 minutes to obtain precipitate A and supernatant A; a portion of the supernatant A is loaded onto an albumin removal centrifugal column, rotated and mixed at room temperature for 15 minutes, and centrifuged at 1000g for 1 minute to remove albumin, thereby obtaining supernatant filtrate A; another portion of the supernatant A is taken and polyethylene glycol is continued to be added to precipitate plasma to obtain a second mixed solution; the second mixed solution is centrifuged at 20,000g for 5 minutes to obtain supernatant B and precipitate B; supernatant B is loaded onto an albumin removal centrifugal column, rotated and mixed at room temperature for 15 minutes, and centrifuged at 1000g for 1 minute to remove albumin, thereby obtaining supernatant filtrate B.

[0019] In some embodiments, in step (2), the volume ratio of the plasma sample to anhydrous acetonitrile is 1:9; the volume ratio of the supernatant filtrate A to trichloroacetic acid is 9:1; the volume ratio of the supernatant filtrate B to trichloroacetic acid is 9:1; and the washing is performed using anhydrous acetonitrile.

[0020] Wherein, in step (2), the precipitate D is washed with anhydrous acetonitrile, and the washing is repeated 3 to 5 times. The single amount of anhydrous acetonitrile is not particularly limited, and is preferably 2.5 times the volume of the supernatant filtrate A; the precipitate E is washed with anhydrous acetonitrile, and the washing is repeated 3 to 5 times. The single amount of anhydrous acetonitrile is not particularly limited, and is preferably 2.5 times the volume of the supernatant filtrate B.

[0021] In some embodiments, in step (3), the precipitate B, the precipitate C, the precipitate D, and the precipitate E are respectively dissolved with a urea solution containing Lys-C enzyme, and after the precipitate is dissolved, a buffer solution and trypsin are added for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, dithiothreitol is added to the enzymatic hydrolyzate of each sample for a reduction reaction, and after the reaction is completed, iodoacetamide is added for an alkylation reaction, and after the reaction is completed, dithiothreitol is added again for a quenching reaction to obtain a peptide sample.

[0022] In some embodiments, in step (3), in the urea solution, the solute is urea, the solvent is 25-50mM, pH=8.4-8.6 TEAB buffer, and the concentration of urea is 8mol / L; in the urea solution containing Lys-C enzyme, the concentration of Lys-C enzyme is 0.020-0.030μg / μL; the amount of Lys-C enzyme used, based on the plasma sample required for each enzymatic hydrolysis sample, the mass volume ratio of the Lys-C enzyme to the plasma sample is 1μg:9μL; the buffer is TEAB buffer, 25-50mM, pH=8.4-8.6; the precipitate B, the precipitate C, the precipitate D, and the precipitate E are respectively dissolved with the urea solution containing Lys-C enzyme, and the buffer is added after the precipitate is dissolved, and the amount of the buffer is to dilute the urea in the system to a final concentration of 2mol / L; the mass ratio of the trypsin to the Lys-C enzyme is 1:1; The enzymolysis is carried out at room temperature for 12-24 hours; the dithiothreitol exists in the form of an aqueous solution, and dithiothreitol is mixed with water to obtain a dithiothreitol aqueous solution; in the system after the dithiothreitol aqueous solution and the enzymolysis solution are mixed, the final concentration of dithiothreitol is 2mmol / L; the reduction reaction is carried out at room temperature and the reaction time is 0.75-1.25h; the iodoacetamide exists in the form of an aqueous solution, and iodoacetamide is mixed with water to obtain an iodoacetamide aqueous solution; in the system after the iodoacetamide aqueous solution and the reaction solution obtained after the reduction reaction are mixed, the final concentration of iodoacetamide is 5mmol / L; the alkylation reaction is carried out at room temperature and the reaction time is 0.5h; in the system after the dithiothreitol aqueous solution and the reaction solution obtained after the alkylation reaction are mixed, the final concentration of dithiothreitol is 5mmol / L; the quenching reaction is carried out at room temperature and the reaction time is 15min.

[0023] In some embodiments, in step (3), in the urea solution, the solute is urea, the solvent is 50mM, pH=8.5 TEAB buffer, and the concentration of urea is 8mol / L; in the urea solution containing Lys-C enzyme, the concentration of Lys-C enzyme is 0.025μg / μL; the amount of Lys-C enzyme used, based on the plasma sample required to start each enzymatic hydrolysis sample, the mass volume ratio of the Lys-C enzyme to the plasma sample is 1μg:9μL; the buffer is TEAB buffer, 50mM, pH=8.5; the precipitate B, the precipitate C, the precipitate D, and the precipitate E are respectively dissolved with the urea solution containing Lys-C enzyme, and the buffer is added after the precipitate is dissolved, and the amount of the buffer is to dilute the urea in the system to a final concentration of 2mol / L; the mass ratio of the trypsin to the Lys-C enzyme is 1:1; the enzymatic hydrolysis, in The enzymolysis is carried out at room temperature for 12-24 hours; the dithiothreitol exists in the form of an aqueous solution, and dithiothreitol is mixed with water to obtain a dithiothreitol aqueous solution; in the system after the dithiothreitol aqueous solution and the enzymatic hydrolysis solution are mixed, the final concentration of dithiothreitol is 2mmol / L; in the reduction reaction, the reaction temperature is room temperature and the reaction time is 1h; the iodoacetamide exists in the form of an aqueous solution, and iodoacetamide is mixed with water to obtain an iodoacetamide aqueous solution; in the system after the iodoacetamide aqueous solution and the reaction solution obtained after the reduction reaction are mixed, the final concentration of iodoacetamide is 5mmol / L; in the alkylation reaction, the reaction temperature is room temperature and the reaction time is 0.5h; in the system after the dithiothreitol aqueous solution and the reaction solution obtained after the alkylation reaction are mixed, the final concentration of dithiothreitol is 5mmol / L; in the quenching reaction, the reaction temperature is room temperature and the reaction time is 15min.

[0024] Therein, the concentration of dithiothreitol in the dithiothreitol aqueous solution is not particularly limited, and is preferably 1 mol / L; the concentration of iodoacetamide in the iodoacetamide aqueous solution is not particularly limited, and is preferably 0.1 mol / L.

[0025] Wherein, the TEAB buffer is Triethylammonium bicarbonate, triethylamine-bicarbonate buffer.

[0026] In some embodiments, in step (4), the desalting column is a C18 desalting column; the desalting cleaning solution is a 0.1 vt% formic acid aqueous solution; the desalting eluent is a 60-65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid;

[0027] The peptide desalting is carried out according to the following steps:

[0028] The peptide sample obtained in step (3) is mixed with an 8-12 vt% trifluoroacetic acid aqueous solution and then loaded onto a desalting column, desalting and washing is performed using a 0.1 vt% formic acid aqueous solution, followed by elution by adding a 60-65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid, collecting the eluate containing the peptide, and drying to obtain the desalted peptide;

[0029] The volume ratio of the peptide sample to the 8-12 vt% trifluoroacetic acid aqueous solution is 9:1; preferably, the peptide sample obtained in step (3) is mixed with the 10 vt% trifluoroacetic acid aqueous solution and then loaded onto a desalting column, desalting and washing is performed using a 0.1 vt% formic acid aqueous solution, and then elution is performed by adding a 65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid, and the eluate containing the peptide is collected and dried to obtain the desalted peptide.

[0030] In some embodiments, preferably, in step (4), the volume ratio of the peptide sample to the 10 vt% trifluoroacetic acid aqueous solution is 9:1.

[0031] Wherein, in step (4), the desalting column is a C18 desalting column, preferably a Sep-Pak C18 desalting column, Waters, Cat#: WAT023590; before use, the desalting column is passed through 100% methanol, and then a 60-65vt% acetonitrile aqueous solution containing 0.1vt% formic acid is added.

[0032] After passing through the column, a 0.1 vt% formic acid aqueous solution without acetonitrile is passed through the column to balance the column; the amount of the 0.1 vt% formic acid aqueous solution is

[0033] Preferably, based on protein, 1 mg of peptide sample requires 1 mL of 0.1 vt% formic acid aqueous solution for desalting; the amount of 60-65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid is preferably, based on protein, 1 mg of peptide sample requires 1 mL of 60-65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid for eluting peptides.

[0034] In some embodiments, in step (5), the desalted peptide obtained in step (4) is subjected to chromatographic fractionation by reverse liquid chromatography; the desalted peptide is dissolved and resuspended in mobile phase A, and then subjected to chromatographic fractionation by a liquid chromatograph, and the peptide fractions obtained are collected and dried;

[0035] The reverse liquid chromatography is performed using a liquid chromatograph, and the specific chromatographic conditions of the liquid chromatograph are as follows:

[0036] Chromatographic column: C18 chromatographic column; fractionation mobile phase A: 8-12 mM ammonium formate aqueous solution, pH = 9.0; fractionation mobile phase B: 90vt% acetonitrile aqueous solution containing 8-12 mM ammonium formate, pH = 9.0; gradient elution, the specific gradient design is as follows: 0.01-15 minutes, 5%-5% mobile phase B; 15-105 minutes, 5-40% mobile phase B; 105-108 minutes, 40-65% mobile phase B; 108-110 minutes, 65-95% mobile phase B; 110-120 minutes, 95-95% mobile phase B; flow rate: 1 mL / min.

[0037] In some embodiments, preferably, in step (5), the desalted peptide obtained in step (4) is subjected to chromatographic fractionation by reverse liquid chromatography; the desalted peptide is dissolved and resuspended in mobile phase A, and then subjected to chromatographic fractionation in a liquid chromatograph, and the peptide fractions obtained are collected and dried;

[0038] The reverse liquid chromatography is performed using a liquid chromatograph, and the specific chromatographic conditions of the liquid chromatograph are as follows:

[0039] Chromatographic column: C18 chromatographic column; fractionation mobile phase A: 10 mM ammonium formate aqueous solution, pH = 9.0; fractionation mobile phase B: 90vt% acetonitrile aqueous solution containing 10 mM ammonium formate, pH = 9.0; gradient elution, the specific gradient design is as follows: 0.01-15 minutes, 5%-5% mobile phase B; 15-105 minutes, 5-40% mobile phase B; 105-108 minutes, 40-65% mobile phase B; 108-110 minutes, 65-95% mobile phase B; 110-120 minutes, 95-95% mobile phase B; flow rate: 1 mL / min.

[0040] In some embodiments, in step (6), the peptide sample collected by fractionation in step (5) is resuspended in a formic acid aqueous solution and injected into a liquid chromatography-mass spectrometer for mass spectrometry detection. The specific chromatographic conditions are as follows:

[0041] Liquid chromatography conditions:

[0042] Column: Reprosil pure C18-AQ, inner diameter 75μm, length 25cm, filled with C18 microspheres, C18 microspheres with a particle size of 1.9μm; column temperature: 50℃; mobile phase A: containing 0.2vt% formic acid, 3vt% DMSO, the balance is deionized water; mobile phase B: 70-80vt% acetonitrile, 0.2vt% formic acid, 3vt% DMSO, the balance is deionized water; gradient elution: 0-80 minutes, 5-25% mobile phase B; 80-110 minutes, 25-40% mobile phase B; 110-115 minutes, 40%-65% mobile phase B; 115-120 minutes, 65%-95% mobile phase B; elution time is 120 minutes; injection flow rate is 0.3μL / min;

[0043] Mass spectrometry conditions:

[0044] The mass spectrometry data were collected in DDA mode with the following parameters: spray voltage 2.1 KV; capillary temperature 320 °C; primary full scan resolution 60,000 at 200 m / z, AGC target: 3e6, Maximum ion time: 50 ms, primary mass scan range 350-1500; Top N: 20; secondary full scan resolution 15,000 at 200 m / z, AGC target: 1e5, Maximum ion time: 105 ms, secondary scan Fixed first mass 100 m / z; fragmentation mode HCD, fragmentation normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s;

[0045] The analysis of mass spectrometry raw data was based on human protein sequences as the database, and the search parameters were as follows: carbamidomethylation on cysteine ​​was set as a fixed modification parameter; oxidation modification on methionine and acetylation modification on the N-terminus of the protein were set as variable modification parameters; trypsin was selected as the specific enzyme cleavage, and the maximum number of missed cleavages allowed for the polypeptide was 2.

[0046] In some embodiments, preferably, in step (6), the peptide sample collected by fractionation in step (5) is resuspended in a 5vt% formic acid aqueous solution and injected into a liquid chromatography-mass spectrometer for mass spectrometry detection. The specific chromatographic conditions are as follows:

[0047] Liquid chromatography conditions:

[0048] Column: Reprosil pure C18-AQ, inner diameter 75μm, length 25cm, filled with C18 microspheres, C18 microspheres with a particle size of 1.9μm; column temperature: 50℃; mobile phase A: containing 0.2vt% formic acid, 3vt% DMSO, the balance is deionized water; mobile phase B: 80vt% acetonitrile, 0.2vt% formic acid, 3vt% DMSO, the balance is deionized water; gradient elution: 0-80 minutes, 5-25% mobile phase B; 80-110 minutes, 25-40% mobile phase B; 110-115 minutes, 40%-65% mobile phase B; 115-120 minutes, 65%-95% mobile phase B; elution time is 120 minutes; injection flow rate is 0.3μL / min;

[0049] Mass spectrometry conditions:

[0050] The mass spectrometry data were collected in DDA mode with the following parameters: spray voltage 2.1 KV; capillary temperature 320 °C; primary full scan resolution 60,000 at 200 m / z, AGC target: 3e6, Maximum ion time: 50 ms, primary mass scan range 350-1500; Top N: 20; secondary full scan resolution 15,000 at 200 m / z, AGC target: 1e5, Maximum ion time: 105 ms, secondary scan Fixed first mass 100 m / z; fragmentation mode HCD, fragmentation normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s;

[0051] The analysis of mass spectrometry raw data was based on human protein sequences as the database, and the search parameters were as follows: carbamidomethylation on cysteine ​​was set as a fixed modification parameter; oxidation modification on methionine and acetylation modification on the N-terminus of the protein were set as variable modification parameters; trypsin was selected as the specific enzyme cleavage, and the maximum number of missed cleavages allowed for the polypeptide was 2.

[0052] In some embodiments, further preferably, in step (6), the mass spectrometry raw data analysis is processed by searching the library using the ThermoProteome Discoverer 2.5 software; the human protein sequences reviewed in the Swiss-Prot protein database are used as the database, and the main search parameters are as follows: carbamidomethylation on cysteine ​​is set as a fixed modification parameter; oxidation modification on methionine and acetylation modification on the N-terminus of the protein are set as variable modification parameters; trypsin is selected as the specific enzyme cleavage, and the maximum number of missed cleavages allowed for the polypeptide is 2.

[0053] Furthermore, the present invention discloses a pre-treatment kit for mass spectrometry detection of proteins in plasma, comprising:

[0054] Surfactant: 8-12% Triton X-100;

[0055] First plasma precipitant: first polyethylene glycol solution;

[0056] Second plasma precipitant: second polyethylene glycol solution;

[0057] Albumin removal centrifugal column: Cibacin blue agarose gel column;

[0058] The first protein precipitation agent: anhydrous acetonitrile;

[0059] Second protein precipitation agent: trichloroacetic acid;

[0060] The third protein precipitation agent: trichloroacetic acid;

[0061] First enzymatic protease: 8 M urea solution containing 0.020-0.030 μg / μL Lys-C enzyme;

[0062] Second enzymatic protease: trypsin;

[0063] Reducing agent: 1 mol / L dithiothreitol aqueous solution;

[0064] Alkylating agent: 0.1 mol / L iodoacetamide aqueous solution;

[0065] Alkylation reaction quenching reagent: 1 mol / L dithiothreitol aqueous solution;

[0066] Desalting column: C18 desalting column;

[0067] Desalting cleaning solution: 0.1vt% formic acid aqueous solution;

[0068] Desalting eluent: 60-65vt% acetonitrile aqueous solution containing 0.1vt% formic acid.

[0069] In some embodiments, the first polyethylene glycol solution, the solute is PEG6000, the solvent is a PBS buffer with a pH of 7.3 to 7.5, and the concentration of PEG6000 is 18 to 22% g / mL; the second polyethylene glycol solution, the solute is PEG6000, the solvent is a PBS buffer with a pH of 7.3 to 7.5, and the concentration of PEG6000 is 30% g / mL; in the urea solution, the solute is urea, and the solvent is a TEAB buffer with a pH of 25-50 mM and a pH of 8.4 to 8.6.

[0070] In some embodiments, preferably, in the first polyethylene glycol solution, the solute is PEG6000, the solvent is a PBS buffer with a pH of 7.4, and the concentration of PEG6000 is 20% g / mL; in the second polyethylene glycol solution, the solute is PEG6000, the solvent is a PBS buffer with a pH of 7.4, and the concentration of PEG6000 is 30% g / mL; in the urea solution, the solute is urea, and the solvent is a TEAB buffer with a pH of 50 mM and a pH of 8.5.

[0071] In some embodiments, preferably, the mass spectrometry detection pre-treatment kit comprises:

[0072] Surfactant: 10% Triton X-100;

[0073] First plasma precipitant: polyethylene glycol solution with a concentration of PEG6000 of 20% g / mL;

[0074] Second plasma precipitant: polyethylene glycol solution with a concentration of PEG6000 of 30% g / mL;

[0075] Albumin removal centrifugal column: Cibacin blue agarose gel column;

[0076] The first protein precipitation agent: anhydrous acetonitrile;

[0077] Second protein precipitation agent: trichloroacetic acid;

[0078] The third protein precipitation agent: trichloroacetic acid;

[0079] First enzymatic protease: 8 M urea solution containing 0.025 μg / μL Lys-C enzyme;

[0080] Second enzymatic protease: trypsin;

[0081] Reducing agent: 1 mol / L dithiothreitol aqueous solution;

[0082] Alkylating agent: 0.1 mol / L iodoacetamide aqueous solution;

[0083] Alkylation reaction quenching reagent: 1 mol / L dithiothreitol aqueous solution;

[0084] Desalting column: C18 desalting column;

[0085] Desalting cleaning solution: 0.1vt% formic acid aqueous solution;

[0086] Desalting eluent: 65 vt acetonitrile in water containing 0.1 vt % formic acid.

[0087] In some embodiments, the mass spectrometry detection pretreatment kit further includes anhydrous acetonitrile, 10vt% trifluoroacetic acid aqueous solution, anhydrous methanol, fractionation mobile phase A, fractionation mobile phase B, 5vt% formic acid aqueous solution, liquid mobile phase A, liquid mobile phase B, and 25-50mM, pH=8.4-8.6 TEAB buffer;

[0088] Preferably, the fractionation mobile phase A is an 8-12 mM ammonium formate aqueous solution, pH = 9.0; the fractionation mobile phase B is a 90 vt% acetonitrile aqueous solution containing 8-12 mM ammonium formate, pH = 9.0; the liquid mobile phase A contains 0.2 vt% formic acid, 3 vt% DMSO, and the remainder is deionized water; the liquid mobile phase B: 70-80 vt% acetonitrile, 0.2 vt% formic acid, 3 vt% DMSO, and the remainder is deionized water.

[0089] In some embodiments, preferably, the mass spectrometry detection pretreatment kit further comprises anhydrous acetonitrile, 10vt% trifluoroacetic acid aqueous solution, anhydrous methanol, fractionation mobile phase A, fractionation mobile phase B, 5vt% formic acid aqueous solution, liquid mobile phase A, liquid mobile phase B, and 50mM, pH=8.5 TEAB buffer;

[0090] Further preferably, the fractionation mobile phase A is a 10mM ammonium formate aqueous solution, pH = 9.0; the fractionation mobile phase B is a 90vt% acetonitrile aqueous solution containing 10mM ammonium formate, pH = 9.0; the liquid mobile phase A contains 0.2vt% formic acid, 3vt% DMSO, and the remainder is deionized water; the liquid mobile phase B: 80vt% acetonitrile, 0.2vt% formic acid, 3vt% DMSO, and the remainder is deionized water.

[0091] Specifically, the high-abundance proteins described in this application refer to proteins with higher content in total protein extracts or plasma, generally including about 20 proteins with the highest concentration, and the concentration range of each protein is about 1 mg / mL. They occupy the vast majority of plasma protein components. High-abundance proteins specifically include albumin, globulin, lipoprotein, complement, coagulation factors, etc.

[0092] Beneficial effects:

[0093] (1) The present invention finds that high-density proteins such as globulins in plasma can be effectively precipitated by polyethylene glycol (PEG6000), and then the albumin component in plasma can be separated by commercially available Cibacron Blue 3G-A, and then the separated components are chromatographed and fractionated by reverse phase liquid chromatography (RPLC) under alkaline pH conditions, and finally the mass spectrometry detection of proteins by liquid chromatography-mass spectrometry can be performed, which can effectively improve the detection depth of proteins in plasma. By this method, the amount of protein detection in plasma can be increased from 2946 to 4073, and the number of protein detection is effectively increased by 38.3%, which greatly improves the detection depth and can provide the possibility for the discovery of clinical disease markers with low abundance in plasma.

[0094] (2) The present invention utilizes PEG6000 to effectively precipitate some high-abundance proteins in plasma. PEG6000 with a final concentration of 10 g / mL% can reduce globulin in plasma from 31.1 g / L to 2.0 g / L; lipoprotein (a) can be reduced from 0.22 g / L to 0.01 g / L; complement C3 and C4 can be reduced from 0.98 g / L and 0.28 g / L to 0.0 g / L, respectively.

[0095] (3) The Cibacron Blue 3G-A column used in the present invention can effectively filter out the albumin in the supernatant after plasma PEG precipitation, and can reduce the albumin in the original plasma from 43.6 mg / mL to 4.8 mg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0097] Figure 1 The figure shows the effect of different concentrations of PEG6000 on the levels of some high-abundance proteins in plasma when the plasma is precipitated.

[0098] Figure 2 The results of SDS-PAGE staining of proteins in different components after plasma precipitation with PEG6000 at different concentrations.

[0099] Figure 3 This is a diagram showing the effect of Cibacron Blue 3G-A agarose gel column on albumin removal; Figure 3 A is a Cibacron Blue 3G-A agarose gel column diagram for filtering out albumin from plasma. Figure 3 B is the SDS-PAGE staining result of the protein.

[0100] Figure 4 The effect of different conditions on the removal of albumin by Cibacron Blue 3G-A agarose gel column; Figure 4 A is the SDS-PAGE staining result diagram for verifying the plasma removal amount of each albumin removal column; Figure 4 B is the determination of the optimal loading amount of plasma precipitated by PEG6000 at a final concentration of 10%; Figure 4 C is the SDS-PAGE staining result of the albumin elution effect of different elution buffers; Figure 4 D is the SDS-PAGE staining result of the effect of polysorbate-20 on the binding of albumin to the column; Figure 4 E is the SDS-PAGE staining result of albumin filtered by repeated use of Cibacron Blue 3G-A agarose gel column; Figure 4 F is the protein separation effect after plasma precipitation with PEG at a final concentration of 20%.

[0101] Figure 5 The main flow chart of the experiment of PEG plasma precipitation and albumin filtration of the present invention.

[0102] Figure 6 This is a correlation diagram of the initial protein detection of PEG-precipitated plasma and albumin after filtration; among them, Figure 6 A is a physical picture of the appearance of each sample during the PEG precipitation plasma and albumin filtration process; Figure 6 B is a graph showing the removal of high-density proteins in each component of plasma extraction by SDS-polyacrylamide gel electrophoresis; Figure 6 C is the LC-MS / MS analysis of plasma samples during the extraction process; Figure 6 D is a diagram showing the differences in protein content in each component of plasma extraction detected by mass spectrometry; Figure 6 E is a graph showing the number of proteins identified in each component of plasma extraction.

[0103] Figure 7 This is a flow chart for detecting and analyzing original plasma and plasma separation components using ultra-deep mass spectrometry detection experimental process.

[0104] Figure 8 The results of ultra-deep protein profiling of plasma and extracted plasma samples; Figure 8 A is a statistical graph of the number of proteins identified in the original plasma and samples after the removal of high-abundance proteins; Figure 8 B is a heat map showing the differences in protein levels detected in each sample.

[0105] Fig. 9It is a data chart of the main plasma protein database or the main research report on the plasma protein group that has been published, as well as a statistical chart of new proteins detected in the sample after PEG precipitation and albumin filtration in the present invention; wherein, Fig. 9 A is the main plasma protein database or the main research report data chart on plasma protein group that has been published. Fig. 9 B is a statistical graph of new proteins detected in the sample after PEG precipitation and albumin removal in the present invention.

[0106] Fig.10 The present invention is a flowchart of the operation steps of the method for pre-treatment of plasma protein by mass spectrometry detection.

[0107] Fig.11 This is a flowchart of the operational steps of the pre-treatment method for ultra-deep mass spectrometry detection of proteins in plasma in "5. Detect and analyze original plasma and three plasma separation components using ultra-deep mass spectrometry detection experimental process". DETAILED DESCRIPTION

[0108] The present invention can be better understood according to the following examples. However, it is easy for those skilled in the art to understand that the contents described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0109] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0110] The Cibacron Blue 3G-A agarose gel column used in the examples of the present invention was purchased from Abcam.

[0111] The polyethylene glycol used in the embodiments of the present invention is polyethylene glycol 6000.

[0112] The "high value" in the "plasma sample with high-value results of protein indicators" described in the embodiments of the present invention means that the detection result of the protein indicator exceeds the upper limit of detection, or exceeds the upper limit of the reference value range of the normal population. For example, the reference range of AFP for normal people is 0-25ng / mL, and the sample with AFP result exceeding 25ng / mL is the AFP high-value sample; similarly, the upper limit of CEA detection is 100U / mL, and the sample with CEA result of ">100.00U / mL" is the CEA high-value sample; similarly, other protein indicators are also judged by referring to the upper limit of detection or the reference value range of the normal population to determine whether they are high values.

[0113] Embodiment 1:

[0114] 1. Determination of PEG concentration used for plasma precipitation

[0115] When using PEG to precipitate plasma, if the concentration is too high, excessive low-abundance proteins will be precipitated together; if the concentration is too low, insufficient protein precipitation will occur. Therefore, in order to determine the appropriate concentration for PEG precipitation of plasma, the present invention uses different concentrations of PEG to precipitate plasma, and determines the appropriate PEG concentration based on the distribution of proteins in the precipitate and the supernatant. The specific experimental steps are as follows:

[0116] (1) Phosphate saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH = 7.4) was used to prepare PEG6000 in PBS buffer with a mass concentration of 4% (w / v), 8% (w / v), 12% (w / v), 16% (w / v), 20% (w / v), and 30% (w / v). The unit of w / v is g / mL, such as 4%, i.e., 4 g of PEG was dissolved in PBS, and the solution was fixed to 100 mL with PBS.

[0117] (2) Take the remaining plasma samples to be discarded after the test of 100 clinical physical examination subjects, mix them, and record them as plasma sample No. 1. Take 180 μL of the plasma sample and add 10% Triton X-100 with a volume of 10% of the plasma to release the proteins in the plasma by rupturing the membrane vesicles in the plasma and reduce the mutual binding between proteins, thereby improving the effect of PEG precipitation and protein separation.

[0118] (3) The mixed system obtained in step (2) was mixed with an equal volume of PBS buffer containing PEG of different concentrations in step (1) to form plasma samples with final PEG concentrations of 2% (w / v), 4% (w / v), 6% (w / v), 8% (w / v), 10% (w / v), and 15% (w / v). In addition, the plasma sample obtained in step (2) was mixed with an equal volume of 20% (w / v) PBS buffer containing PEG obtained in step (1), and then mixed with an equal volume of 30% (w / v) PBS buffer containing PEG to form a plasma sample with a final PEG concentration of 20% (w / v). The mixture was mixed by oscillation, and then rotated and shaken at room temperature for 10 minutes. Centrifuged at 20,000g for 5 minutes. The supernatant was retained and the precipitate was dissolved with RIPA buffer (25mM Tris-HCl, pH 7.6, 125mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) in the initial plasma volume. Wherein, the unit of w / v is g / mL.

[0119] (4) The collected supernatant, dissolved precipitate and original plasma were subjected to routine biochemical tests in the clinical laboratory (hospital laboratory) to determine the levels of protein indicators, including total protein, albumin, globulin, apolipoprotein AI, apolipoprotein B, lipoprotein (a) and complement C3 and C4. The results are as follows: Figure 1 As shown in the figure, except for albumin and apolipoprotein AI, when the final concentration of PEG in plasma is 10%, most of the proteins have been precipitated. It can be seen from the figure that PEG6000 can effectively precipitate some high-abundance proteins in plasma. Among them, PEG6000 with a final concentration of 10g / mL% can reduce the globulin in plasma from 31.1g / L to 2.0g / L; lipoprotein (a) can be reduced from 0.22g / L to 0.01g / L; complement C3 and C4 can be reduced from 0.98g / L and 0.28g / L to 0.0g / L respectively.

[0120] (5) Take these samples separately for SDS-PAGE (polyacrylamide gel electrophoresis) and Coomassie brilliant blue staining. Take 10 μL of each of the above samples and dilute them with 1xLDS loading buffer (2% Lithium Dodecyl Sulfate, 10% glycerol, 0.2Mtriethanolamine-HCl, pH 7.6, 0.025% phenol red, 0.025% Coomassie G250) so that all samples are diluted 20 times relative to their concentration in the original plasma. For example, the supernatant sample containing a final PEG concentration of 2% (w / v) has been diluted equally with the PBS buffer containing PEG, so it is diluted 10 times with 1xLDS (i.e., 90 μL is enough). All the precipitate samples obtained in step (3) are first dissolved with 8M urea (prepared in PBS) with a volume of 4 times the initial plasma volume, and then 10 μL is taken and 40 μL of 1xLDS is added to make a 5-fold dilution. DTT (Dithiothreitol) was added to each sample at a final concentration of 10 mM and heated at 75°C for 10 minutes. 5 μL of each sample was added to a 4-12% precast polyacrylamide gel and SDS-PAGE (200 V, 45 min) was performed in MOPS (3-morpholinopropanesulfonic acid) electrophoresis buffer. The samples were then stained with Coomassie brilliant blue solution (0.1% Coomassie R-250, 40% ethanol, 10% acetic acid) for half an hour and then destained (10% ethanol and 7.5% acetic acid).

[0121] The changes of high-abundance proteins in plasma after plasma precipitation with polyethylene glycol (PEG6000) at different concentrations are shown in the figure Figure 2 As shown, SDS-polyacrylamide gel electrophoresis shows the precipitation of high-abundance plasma proteins under precipitation with various concentrations of PEG6000. The higher the PEG concentration, the more precipitation there is.

[0122] According to the above biochemical protein detection and SDS-PAGE staining results, a final concentration of 10% (w / v) PEG is a more appropriate concentration, wherein the unit of w / v is g / mL.

[0123] (6) Analyze the effect of PEG precipitation at a final concentration of 10% (w / v) or above on the target protein.

[0124] Among the remaining plasma samples tested in the clinical laboratory, some plasma samples with high-value results for protein indicators are selected and mixed, which is recorded as plasma sample No. 2.

[0125] The plasma sample No. 2 was mixed with PBS buffer containing PEG6000 at a concentration of 20% (w / v) and 30% (w / v), respectively, and the PEG-precipitated plasma samples with a final PEG6000 concentration of 10% (w / v), 15% (w / v) and 20% (w / v) were prepared according to the method in step (3) above, and the mixture was vortexed and then rotated and shaken at room temperature for 10 minutes. Centrifuged at 20,000 g for 5 minutes. The supernatant was retained, and each supernatant and the original plasma (as the initial control) were tested for common tumor markers in the clinical laboratory of Gulou Hospital, including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), carbohydrate antigen 125 (CA125), ferritin (FER), carbohydrate antigen 19-9 (CA19-9, i.e. CA199 in Table 1), carbohydrate antigen 15-3 (CA15-3), carbohydrate antigen 72-4 (CA72-4), carbohydrate antigen 242 (CA242), carbohydrate antigen 50 (CA50), cytokeratin 19 fragment (CYFRA21-1), neuron-specific enolase (NSE), total prostate-specific antigen (TPSA), human epididymis protein 4 (HE4), squamous cell carcinoma antigen (SCCA), and pro-gastrin peptide (Pro-GRP). The specific results are shown in Table 1 below. It can be seen from the table that PEG precipitation has little effect on the levels of these indicators, and all of them disappeared without precipitation.

[0126] Table 1 Effects of PEG precipitation at 10% and above on the commonly used clinical protein indicators in plasma

[0127]

[0128] 2. Exploring the conditions for removing albumin from plasma using Cibacron blue dye agarose resin

[0129] The present invention utilizes Abcam's Cibacron Blue 3G-A agarose gel column ( Figure 3 A) Albumin in plasma is filtered.

[0130] Unless otherwise specified in this experiment, the composition of the PBS buffer used was: phosphate saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH = 7.4).

[0131] The PBS buffer containing 2M NaCl (sodium chloride) used in this experiment was prepared according to the following steps: NaCl was added to phosphate (PBS) buffer (10mM Na2HPO4, 1.8mM KH2PO4, 137mM NaCl, 2.7mM KCl, pH=7.4) to make the final concentration of NaCl 2M.

[0132] The specific experimental process is as follows:

[0133] (1) Take a Cibacron Blue 3G-A agarose gel column and wash the column twice with 400 μL PBS to equilibrate the column before loading the sample;

[0134] (2) Mix and dilute 100 μL of plasma sample No. 1 with 300 μL of PBS buffer;

[0135] (3) Add the diluted plasma sample to the equilibrated column and rotate to mix at room temperature for 15 minutes;

[0136] (4) Centrifuge at 1000 g for 1 min and collect the filtrate (denoted as Flow-through, FL);

[0137] (5) Wash the column twice with 400 μL PBS;

[0138] (6) Add 400 μL of PBS buffer containing 2 M NaCl (sodium chloride) to the column and rotate to mix at room temperature for 15 minutes;

[0139] (7) Centrifuge at 1000 g for 1 min and collect the eluate (denoted as Eluted);

[0140] (8) The filtrate, eluate and original plasma were subjected to SDS-PAGE and Coomassie Brilliant Blue staining to observe the deproteinization effect.

[0141] SDS-PAGE staining results are shown in Figure Figure 3B. As can be seen from the figure, the Cibacron Blue 3G-A agarose gel column can effectively remove albumin from plasma, and other proteins are not significantly affected.

[0142] 3. Clarify the effect of different experimental conditions on the removal of albumin from plasma by Cibacron blue dye agarose resin

[0143] Unless otherwise specified in this experiment, the composition of the PBS buffer used was: phosphate saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH = 7.4).

[0144] The PBS buffer containing 2M NaCl (sodium chloride) used in this experiment was prepared according to the following steps: NaCl was added to phosphate (PBS) buffer (10mM Na2HPO4, 1.8mM KH2PO4, 137mM NaCl, 2.7mM KCl, pH=7.4) to make the final concentration of NaCl 2M.

[0145] The markers used in this experiment are the same.

[0146] (1) Determine the albumin removal capacity of each filtration column, that is, the appropriate amount of plasma loaded. If the amount of sample loaded is too much, the albumin in it cannot be fully removed. If the amount of sample loaded is too little, target proteins other than albumin will also be lost due to nonspecific adsorption to the column.

[0147] Add 100 μL of plasma sample No. 1 and 300 μL of PBS to the Cibacron Blue 3G-A agarose gel column and rotate and mix at room temperature for 15 minutes to allow the albumin in the sample to fully bind to the Cibacron Blue 3G-A agarose gel column. Repeat steps (4), (5), (6), and (7) in "2. Exploring the conditions for removing albumin from plasma using Cibacron blue dye agarose resin", collect the filtrate (denoted as FL1), and collect the eluate (denoted as E1). Subsequently, after re-equilibrating the column with PBS, the collected 400 μL filtrate FL1 was added to the column and mixed by rotation at room temperature for 15 minutes. Then, the steps (4), (5), (6), and (7) in "2. Exploring the conditions for removing albumin from plasma using Cibacron blue dye agarose resin" were repeated to collect the filtrate (recorded as FL2) and elute the collected eluate (recorded as E2). Subsequently, after re-equilibrating the column with PBS, the collected 400 μL filtrate FL2 was passed through the column and collected, eluted, and equilibrated according to the above method. This was repeated 5 times, and FL1, FL2, FL3, FL4, FL5 and E1, E2, E3, E4, and E5 were collected in turn. These samples and the original plasma were subjected to SDS-PAGE, and the distribution of albumin in each sample was observed.

[0148] If the column can bind all the albumin in 100 μL of plasma, then we should see that albumin is mainly concentrated in E1 and decreases significantly from E2. At the same time, albumin should decrease significantly from FL1 to FL2, and the results show that this is indeed the case (such as Figure 4 This indicates that the albumin removal capacity of Cibacron Blue 3G-A agarose gel column is about 100 μL plasma. One Cibacron Blue 3G-A agarose gel column can fully remove the albumin in 100 μL plasma.

[0149] (2) Determine the amount of albumin removed from plasma by Cibacron Blue 3G-A agarose gel column after PEG precipitation

[0150] The PBS buffer containing 20% ​​(w / v) PEG prepared in step (1) under "1. Determination of PEG concentration for plasma precipitation" was used in this experiment, hereinafter referred to as 20% PEG, wherein w / v is g / mL.

[0151] The plasma sample No. 1 was mixed with an equal volume of 20% PEG and precipitated to obtain a plasma sample with a final PEG concentration of 10%. The sample was centrifuged to obtain a precipitate and a supernatant, and the precipitate was recorded as 10% PEG pellet; then 400μL, 300μL, 200μL, and 100μL of the supernatant sample were taken respectively, and the supernatant was filtered through Cibacron Blue 3G-A agarose gel, and the filtrate sample (filtrate FL) was collected. The filtrate sample FL obtained through the column was subjected to SDS-PAGE with the original plasma sample and the 10% PEG pellet.

[0152] The results are as follows Figure 4 As shown in Figure B, it can be seen that starting from the filtrate FL sample obtained by filtering 200 μL of supernatant, the albumin has basically disappeared, which shows that 200 μL of supernatant sample (that is, using the initial 100 μL of plasma) is the appropriate sample volume for the column.

[0153] (3) Elution effect of different elution buffers

[0154] Since the eluted samples may be subjected to mass analysis and mass spectrometry detection, it is necessary to use reagents that are compatible with downstream analysis for elution. This experiment is to determine whether these reagents have an elution effect.

[0155] The RIPA (radioimmunoprecipitation assay) buffer used in this experiment was: 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate.

[0156] The supernatant of 200 μL of plasma sample (starting from 100 μL of plasma sample No. 1) precipitated with PEG at a final concentration of 10% prepared according to the method in "(2) Determining the amount of albumin removed by filtration through Cibacron Blue 3G-A agarose gel column after plasma PEG precipitation" was passed through a Cibacron Blue 3G-A agarose gel column after centrifugation, rotated and mixed at room temperature for 15 minutes, centrifuged at 1000 g for 1 minute, and the filtrate was collected (denoted as Flow-through, FL). The column was washed twice with 400 μL PBS (the obtained sample was denoted as Washed by binding buffer), and then eluted with 400 μL RIPA and 1M, 2M, 4M, and 8M urea solutions (Urea) prepared in PBS, respectively, to obtain different eluates. Finally, 400 μL of the eluate in the Albumin Depletion Kit (ab241023) was used for elution and collection (the obtained sample was denoted as elution buffer).

[0157] The original plasma, filtrate FL, the eluate obtained by elution with the binding buffer, the eluate obtained by elution with the elution buffer, RIPA, and the eluates obtained by elution with 1M, 2M, 4M, and 8M urea (Urea) were subjected to SDS-PAGE.

[0158] Specific results such as Figure 4 As shown in Figure C, both RIPA and urea have elution effects, so both can be considered to replace the elution solution in the kit so that downstream experiments can be smoothly connected. RIPA solution can elute the bound protein on the column after filtration, which also means that the loaded plasma sample cannot contain RIPA components, otherwise it will affect the binding of albumin to the column.

[0159] (4) Determine the effect of polysorbate 20 (Tween-20) on albumin binding to the column

[0160] Tween-20 is a commonly used detergent in biological experimental solutions, which can effectively prevent non-specific binding of proteins. If used in plasma, it can effectively prevent the target protein in plasma from being lost through non-specific adsorption to the column, but whether Tween-20 affects the binding of albumin to the column needs to be determined.

[0161] According to the method in "(2) Determining the amount of albumin removed by Cibacron Blue 3G-A agarose gel column after plasma PEG precipitation", a plasma sample with a final concentration of 10% PEG precipitation was prepared. Multiple 200 μL portions of the plasma system with a final concentration of 10% PEG precipitation (using plasma sample No. 1) were centrifuged to obtain a precipitate (referred to as 10% PEG precipitation) and a supernatant (referred to as 10% PEG supernatant). 0.1%, 0.25%, 0.5%, and 1% Tween-20 were added to the supernatant, respectively, and a control group without Tween-20 was used (0%). 200 μL PBS was added to increase the total volume to 400 μL. Then, albumin was removed by Cibacron Blue 3G-A agarose gel column, centrifuged at 1000 g for 1 min, and the filtrate (FL) was collected. Each FL sample was collected and subjected to SDS-PAGE with the original plasma, 10% PEG precipitation, and 10% PEG supernatant samples. After Coomassie staining, the results are as follows Figure 4 As shown in D, Tween-20 has no effect on the binding of albumin to the column. Note: The amount of Tween-20 used is the volume fraction, vol / vol, such as 0.1%, which means 1mL 100% Tween-20 is dissolved in 1L water.

[0162] (5) Determine whether the Cibacron Blue 3G-A agarose gel column can be reused

[0163] Because albumin and Cibacron Blue on Cibacron Blue 3G-A agarose gel column are mainly attracted by electrostatic attraction. After binding, the protein bound to it can be fully eluted by elution with high concentration salt containing 2M NaCl (sodium chloride). If it is filtered and washed with a column binding buffer without high salt, the column can be effectively balanced and used again. This can effectively reduce costs when filtering the same plasma and processed samples repeatedly in preliminary experiments.

[0164] In the experiment, a Cibacron Blue 3G-A agarose gel column was taken, 100 μL of plasma sample No. 1 was added, and steps (4) to (7) in "2. Exploring the conditions for removing albumin from plasma using Cibacron blue dye agarose resin" were repeated, and the filtrate (FL) was collected by centrifugal filtration, and the eluate (Eluted) was collected after elution, and equilibrated; then, a new 100 μL of plasma was added, and the above steps were repeated, and the filtrate (FL) was collected by centrifugal filtration, and the eluate (Eluted) was collected after elution, and equilibrated; this was repeated 5 times, and the filtrate (FL) and the eluate (Eluted) were collected separately, and then SDS-PAGE and Coomassie staining were performed.

[0165] The results are as follows Figure 4 As shown in Figure E, it can be seen that the repeated use of the Cibacron Blue 3G-A agarose gel column for 5 times has no obvious effect on the filtration of albumin, indicating that the Cibacron Blue 3G-A agarose gel column can be used repeatedly for at least 5 times.

[0166] (6) Testing the effect of protein separation in the entire experimental process of plasma PEG precipitation and supernatant column filtering for albumin removal

[0167] If each process is effective for protein separation, the protein bands in each separated sample should be clearly distinguished from the protein bands in other separated samples (if the separation effect is not good, the protein bands will be slightly different from the samples before and after separation, indicating no separation effect).

[0168] Because according to Figure 1 The data speculation and Figure 2The results show that the precipitation of high-abundance proteins in plasma by PEG in the PEG-precipitated plasma sample with a final concentration of 20% is more thorough, so the effect will be better. Therefore, this experiment uses the method under "1. Determination of PEG concentration used for precipitated plasma" to prepare 90 μL of No. 1 plasma specimen into 400 μL of PEG-precipitated plasma system with a final PEG concentration of 20% (w / v) (w / v unit is g / mL), centrifuge to obtain a precipitate (denoted as 20% PEG / pellet) and a supernatant (denoted as 20% PEG / Sup), then load the supernatant onto a Cibacron Blue 3G-A agarose gel column, rotate and mix at room temperature for 15 minutes, centrifuge at 1000 g for 1 min, collect the filtrate (denoted as 20% PEG / Sup / FL), and use 400 μL The column was washed twice with PBS, 400 μL of PBS buffer containing 2 M NaCl (sodium chloride) was added to the column, and the column was rotated and mixed at room temperature for 15 minutes. The column was centrifuged at 1000 g for 1 minute, and the eluate was collected (denoted as 20% PEG / Sup / Eluted).

[0169] The original plasma, 20% PEG / pellet, 20% PEG / Sup, 20% PEG / Sup / FL, and 20% PEG / Sup / Eluted were subjected to SDS-PAGE and Coomassie Brilliant Blue staining to observe the protein separation effect. Figure 4 As shown in Figure F, it can be seen that the simple proteins of each separated sample are obviously different, and are consistent with expectations: except for albumin, most other high-abundance proteins visible to the naked eye are effectively precipitated in 20% PEG / pellet; and the albumin in the supernatant of 20% PEG / Sup is almost completely filtered out after passing through the Cibacron blue gel column.

[0170] 4. Preliminary determination of the effect of mass spectrometry after plasma separation using PEG precipitation and Cibacron blue dye agarose resin

[0171] Unless otherwise specified in this experiment, the composition of the PBS buffer used was: phosphate saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH = 7.4).

[0172] The PBS buffer containing 2M NaCl (sodium chloride) used in this experiment was prepared according to the following steps: NaCl was added to phosphate (PBS) buffer (10mM Na2HPO4, 1.8mM KH2PO4, 137mM NaCl, 2.7mM KCl, pH=7.4) to make the final concentration of NaCl 2M.

[0173] (1) Separation of plasma by PEG precipitation and centrifugation of the supernatant through a column

[0174] 10 μL of 10% Triton X-100 (final concentration 1%) was added to 90 μL of plasma sample No. 1, and then mixed with PBS buffer containing 20% ​​(w / v) PEG6000 (w / v unit is g / mL) in equal volumes to form a PEG6000 precipitation plasma system with a final concentration of 10% (w / v). After mixing at room temperature for 10 minutes, centrifuge at 20,000g for 5 minutes to collect the precipitate (referred to as 10% PEG / pellet) and the supernatant (referred to as 10% PEG / sup). The 10% PEG / sup supernatant was then mixed with PBS buffer containing 30% (w / v) PEG in equal volumes to form a PEG precipitation plasma system with a final concentration of 20% (w / v). After mixing for 10 minutes, centrifuge at 20,000g for 5 minutes to collect the precipitate (referred to as 20% PEG / pellet) and the supernatant (referred to as 20% PEG / sup). Load the 20% PEG / sup supernatant onto a Cibacron Blue 3G-A agarose gel column, rotate and mix at room temperature for 15 minutes, centrifuge at 1000g for 1min, collect the filtrate (recorded as 20% PEG / sup / FL), wash the column twice with 400μL PBS, add 400μL PBS buffer containing 2M NaCl (sodium chloride) to the column, rotate and mix at room temperature for 15 minutes, centrifuge at 1000g for 1min, and collect the eluate (recorded as Eluted). See the detailed process for details. Figure 5 .

[0175] (2) The appearance of each separated component sample in step (1) and the protein distribution after SDS-PAGE were recorded. For details, see Figure 6 A. Figure 6 B.

[0176] (3) The changes in the levels of protein indicators such as commonly used clinical tumor markers in the samples before and after the final extraction are shown in Table 2. The remaining plasma samples with high values ​​of protein indicators in the clinical laboratory test of Gulou Hospital were taken and mixed, which was recorded as plasma sample No. 3. Since the high-value samples will exceed the detection limit of the detection method. In this experiment, all high-value specimens were diluted, and then the absolute values ​​of the test results were obtained to compare the results before and after the extraction. The plasma sample No. 3 was mixed with PBS buffer containing PEG6000 to prepare a PEG precipitated plasma sample with a final PEG6000 concentration of 20% g / mL, centrifuged to obtain the supernatant, and the supernatant was passed through a Cibacron Blue 3G-A agarose gel column to obtain 20% PEG / Sup / FL, which was recorded as the post-extraction sample. The sample before the extraction was the original plasma sample. In Table 2, Total PSA is (total prostate-specific antigen), FreePSA is (free prostate-specific antigen), PAP is (prostatic acid phosphatase), β2-microglobulin is (β2-microglobulin), Total protein is total protein, Albumin is albumin, lgG is (immunoglobulin G), lgA is (immunoglobulin A), and lgM is (immunoglobulin M). As can be seen from the table, the Cibacron Blue 3G-A gel column can effectively filter out albumin in the supernatant after plasma PEG precipitation, and can reduce the albumin in the original plasma from 43.6 mg / mL to 4.8 mg / mL. Among them, the enrichment multiple in the table refers to the change in the concentration of the sample detection index before and after extraction relative to the change in the total protein of the sample before and after extraction. For example, the enrichment multiple after AFP extraction is: (4295.2 / 10.8) / (8253.2 / 65.6)=3.16.

[0177] Table 2 Changes in the content of plasma protein indicators before and after plasma extraction

[0178]

[0179] (4) Mass spectrometry was performed on the proteins in each separated sample in “(1) separating plasma by PEG precipitation of plasma and centrifugation of the supernatant and then passing it through a column”. The specific experimental process is as follows:

[0180] ① Dissolve the PEG precipitated samples (10% PEG / pellet and 20% PEG / pellet, respectively) in 8M urea solution (prepared with PBS as solvent) at 3 times the volume of the starting plasma overnight (protein concentration is about 5-10 μg / μL), and add 1 / 3 volume of 4xLDS protein loading buffer to 1x;

[0181] The original plasma was diluted to 20 times the plasma volume in 1x LDS (protein concentration was approximately 3-4 μg / μL);

[0182] After PEG precipitation of plasma, the supernatant (20% PEG / sup), 20% PEG / sup / FL, and Eluted samples were directly added with 1 / 3 volume of 4xLDS to 1x (protein concentration is about 2-8μg / μL). Note: The protein concentration was obtained by BCA quantitative analysis.

[0183] ②Reductive alkylation of samples. Take 20-50 μL of each sample in ① (protein concentration is about 2-10 μg / μL), add 1M TEAB aqueous solution to a final TEAB concentration of 50 mM to ensure that the pH is around 8.5; then add 1M DTT aqueous solution to a final DTT concentration of 3 mM, and react at 75°C for 10 minutes to fully reduce the sample. Then cool on ice, add 0.1M IAA aqueous solution (iodoacetamide aqueous solution) to a final IAA concentration of 10 mM, stir and react at room temperature in the dark for 15 minutes to obtain the reduced alkylated sample.

[0184] ③ Gel electrophoresis of samples. Select 4-12% polyacrylamide gel, try to select 10 channels, add 50μg of each processed sample in ②, perform SDS-PAGE, and perform electrophoresis at 200V. When all samples run into the lanes and separate, stop electrophoresis (3-5 minutes), remove the gel and perform Coomassie staining. When the protein is visible in the staining (10-30 minutes), cut out the gel blocks where the protein is located (about 0.5cm long).

[0185] ④ Enzymatic hydrolysis of sample protein in gel block. Cut each gel block into 1mm 3 . Decolorize the gel block with 25 mM TEAB aqueous solution (pH=8.5) containing 25 vt.% acetonitrile for at least 3-5 times to remove the Coomassie brilliant blue pigment and impurities such as LDS remaining in the gel block. Use 100% acetonitrile to drain the water from the gel block and drain it on a drainer. Add an appropriate amount of 25 mM TEAB aqueous solution (pH=8.5) containing 10 ng / μL trypsin (Promega, Sequencing Grade Modified Trypsin, Frozen, Cat. #V5113) to the drained gel block and wait on ice for it to fully soak and swell (0.5-2 hours). During this period, add a small amount of 25 mM TEAB aqueous solution (pH=8.5) containing 10 ng / μL trypsin at any time according to the swelling of the gel block, so that there is basically no excess liquid on the surface of the gel block. After the gel block is fully expanded (0.5-2 hours), add about 20 μL of 25 mM TEAB aqueous solution (pH=8.5) and move to 37°C for enzyme digestion for 24-48 hours.

[0186] ⑤ Extraction of peptides from the gel block after enzymatic hydrolysis. Centrifuge the sample obtained in ④ at 20,000 g for 1 minute, and aspirate the liquid sample outside the gel block. Add 25 μL of 50 vt% acetonitrile aqueous solution containing 5 vt% formic acid to the gel block, centrifuge it twice, aspirate the liquid sample outside the gel block, and merge it into the previously aspirated liquid sample; then add 100 μL of 100% acetonitrile, thoroughly extract all liquid samples, and merge them with the previous samples; drain the extracted sample liquid on a drainer to obtain a peptide sample.

[0187] ⑥ LC-MS / MS mass spectrometry detection of peptides. About 1 μg of each peptide fraction sample collected above was resuspended in 5vt% formic acid aqueous solution, and then loaded onto a mass spectrometer (Thermo Q Exactive HF-X) for detection. The mass spectrometer was connected in series with a nanoliter liquid chromatography system (Thermo EASY-nLC 1200).

[0188] Liquid chromatography conditions:

[0189] Laboratory-made chromatographic column: Reprosil pure C18-AQ, inner diameter 75μm, length 25cm, filled with C18 microspheres, C18 microspheres with a particle size of 1.9μm; column temperature: 50°C; mobile phase A: contains 0.2vt% formic acid, 3vt% DMSO, and the balance is deionized water; mobile phase B: 80vt% acetonitrile, 0.2vt% formic acid, 3vt% DMSO, and the balance is deionized water; gradient elution: B phase 5-25% (0-80 minutes), B phase 25-40% (80-110 minutes), B phase 40%-65% (110-115 minutes), B phase 65%-95% (115-120 minutes); elution time is 120 minutes; injection flow rate is 0.3μL / min.

[0190] Mass spectrometry conditions:

[0191] The mass spectrometry data were collected in DDA mode of Thermo Q Exactive_HF-X with the following parameters: spray voltage 2.1 KV; capillary temperature 320 °C; primary full scan resolution 60,000 (at 200 m / z), AGC target: 3e6, Maximum ion time: 50 ms, primary mass scan range 350-1500; Top N: 20; secondary full scan resolution 15,000 (at 200 m / z), AGC target: 1e5, Maximum ion time: 105 ms, secondary scan Fixed firstmass 100 m / z; fragmentation mode HCD, fragmentation normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s.

[0192] ⑦ Retrieval of peptides. The analysis of mass spectrometry raw data was processed by searching the database using Thermo Proteome Discoverer 2.5 software. The human protein sequences reviewed in the Swiss-Prot protein database were used as the database. The main search parameters were as follows: carbamidomethyl on cysteine ​​was set as a fixed modification parameter; oxidative modification on methionine and acetylation modification on the N-terminus of the protein were set as variable modification parameters. Trypsin was selected for specific enzyme cleavage, and the maximum number of missed cleavages allowed for the peptide was 2.

[0193] (5) Test results. The LC spectra of the six experimental samples (original plasma, 10% PEG / pellet, 20% PEG / pellet, 20% PEG / sup, 20% PEG / sup / FL, Eluted) analyzed by mass spectrometer showed great differences among them (e.g. Figure 6 C), indicating the effective separation of each component sample. The protein levels in each component sample were compared by mass spectrometry, and the results showed that there were large differences between them (such as Figure 6 D). In terms of the number of proteins, 348 proteins were detected when the plasma sample was directly tested; 342, 322, 181, 339, and 81 proteins were identified in the remaining five component samples, respectively. After removing the commonly detected proteins, a total of 655 proteins were detected in the five component samples. Together with the proteins in the original plasma, a total of 694 proteins were detected. This result shows that the number of proteins detected in the original plasma can be increased from 348 to 694 after PEG precipitation of plasma and filtration through an albumin-free column, which greatly increases the number of plasma proteins detected. For details, see Figure 6 E.

[0194] 5. Use ultra-deep mass spectrometry to detect and analyze the original plasma and three plasma separation components

[0195] This method mainly uses reverse phase liquid chromatography (RPLC) under alkaline pH conditions to perform chromatographic fractionation on peptide samples. The peptides in the sample are gradually eluted by chromatography according to the size of the hydrophobicity and collected in about 100 tubes in turn. Then the peptide samples in each tube are tested by LC-MS / MS. The core technology of this experimental process is to fractionate the samples that were originally tested on the machine at one time into about 100 samples through RPLC and then test them one by one to improve the detection depth.

[0196] Unless otherwise specified in this experiment, the composition of the PBS buffer used was: phosphate saline (PBS) buffer (10 mM Na2HPO4, 1.8 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, pH = 7.4).

[0197] The PBS buffer containing 2M NaCl (sodium chloride) used in this experiment was prepared according to the following steps: NaCl was added to phosphate (PBS) buffer (10mM Na2HPO4, 1.8mM KH2PO4, 137mM NaCl, 2.7mM KCl, pH=7.4) to make the final concentration of NaCl 2M.

[0198] The reagents used in this experiment are shown in Table 3:

[0199] Table 3

[0200]

[0201] (1) Experimental sample selection

[0202] 25 μL of 10% Triton X-100 (final concentration of 1%) was added to 225 μL of plasma sample No. 1 from a healthy person, and then mixed with an equal volume of PBS buffer (w / v in g / mL) containing 20% ​​(w / v) PEG6000 to form a PEG precipitation plasma system with a final concentration of 10% (w / v). After mixing at room temperature for 10 minutes, centrifuge at 20,000g for 5 minutes, and collect the precipitate (referred to as 10% PEG / pellet) and the supernatant (10% PEG / sup). Take 200 μL of the 10% PEG / sup supernatant and load it onto a Cibacron Blue3G-A agarose gel column, rotate and mix at room temperature for 15 minutes, centrifuge at 1000g for 1 minute, and collect the filtrate (referred to as 10% PEG / sup / FL). Take another 200 μL of 10% PEG / sup supernatant and mix it with an equal volume of PBS buffer containing 30% (w / v) PEG6000 to form a PEG precipitation plasma system with a final concentration of 20% (w / v). After mixing for 10 minutes, centrifuge at 20,000g for 5 minutes to collect the precipitate (recorded as 20% PEG / pellet) and the supernatant (recorded as 20% PEG / sup). Load the 20% PEG / sup supernatant onto the Cibacron Blue 3G-A agarose gel column, rotate and mix at room temperature for 15 minutes, centrifuge at 1000g for 1 minute, and collect the filtrate (recorded as 20% PEG / sup / FL).

[0203] Original plasma, 20% PEG / pellet, 10% PEG / sup / FL and 20% PEG / sup / FL. Among them, 10% PEG / sup / FL is obtained by precipitating plasma with PEG at a final concentration of 10%, and then filtering out albumin through a Cibacron Blue 3G-A agarose gel column. The protein concentration in each sample was estimated based on the aforementioned BCA quantitative results (the protein concentrations in the above four samples were 75.5μg / μL, 10.1μg / μL, 16.5μg / μL, and 7.9μg / μL, respectively), and about 4mg of protein was taken from each sample. For the specific experimental process, please see the above section. For the flowchart of this experiment using polyethylene glycol to precipitate original plasma and remove albumin, see Fig.11 .

[0204] (2) Sample processing:

[0205] The 20% PEG / pellet was directly used for proteolysis in step (3) without post-treatment. The original plasma sample was mixed with anhydrous acetonitrile for precipitation (after the plasma sample was mixed with acetonitrile, the volume fraction of acetonitrile was 90%), and the precipitate was collected as a sample.

[0206] The 10% PEG / sup / FL and 20% PEG / sup / FL samples were precipitated with TCA (Trichloroacetic acid)-acetonitrile. The specific experimental process is as follows:

[0207] ① Add 1 / 9 volume of 100% TCA (TCA final volume concentration is 10%) to the pre-cooled liquid sample (10% PEG / sup / FL or 20% PEG / sup / FL) on ice, shake and mix to fully precipitate the protein, and place on ice for half an hour;

[0208] ② Centrifuge at 20,000 g for 10 minutes;

[0209] ③Discard the supernatant, wash the precipitate with 500 μL of anhydrous acetonitrile, centrifuge at 20,000 g for 10 minutes, repeat 3 times, and finally keep the precipitate sample for later use.

[0210] (3) Proteolysis and reductive alkylation:

[0211] After each treatment, 400 μL of 10 μg Lys-C enzyme (Lysyl Mass Spectrometry Grade, Wako, Cat#: 125-05061) 8M urea solution (prepared with 50mM pH 8.5TEAB), rotate at room temperature for 2-4 hours until the precipitate dissolves; then dilute the urea to 2M with 1200μL of 50mM TEAB (pH 8.5), add 10μg trypsin to each sample, and continue to rotate at room temperature overnight for 12-24 hours. The next day, add 1M DTT aqueous solution to each sample to a final concentration of 2mM DTT, react at room temperature for 1 hour, add 0.1M IAA aqueous solution to a final concentration of 5mM IAA, react at room temperature for half an hour, then add 1M DTT aqueous solution to a final concentration of 5mM DTT, react at room temperature for 15 minutes (to quench the remaining IAA), and obtain.

[0212] (4) Desalting of sample peptides:

[0213] ① Add 1 / 9 volume of 10 vt% TFA aqueous solution to the four peptide samples obtained in the above "(3) proteolysis" to make the final volume concentration of TFA (trifluoroacetic acid) be 1 vt%;

[0214] ② Take SPE desalting column (Sep-Pak C18 desalting column, Waters, Cat#: WAT023590);

[0215] ③ After passing 1 mL of 100% methanol through the column, add 1 mL of 65vt% acetonitrile aqueous solution containing 0.1vt% formic acid, and then pass 3 mL of 0.1vt% formic acid aqueous solution without acetonitrile through the column to balance the column;

[0216] ④ Take 1 mg of peptide sample in terms of protein from each sample, add it to the column, and pass it through the column;

[0217] ⑤ Wash the column with 1 mL 0.1vt% FA aqueous solution (formic acid aqueous solution);

[0218] ⑥ Add 1 mL of 65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid and pass through the column, and collect the eluted peptides;

[0219] ⑦ Repeat the above ③-⑥ to desalt the remaining 3 mg of peptide sample in terms of protein in the sample;

[0220] ⑧ Combine all peptides eluted after desalting and drain to obtain desalted peptides.

[0221] (5) RPLC fractionation of the desalted peptide fragments under alkaline pH conditions:

[0222] A liquid chromatography system (Shimadzu) and a C18 column (XBridge C18 column 3.5um, 4.6mm x250mm, Waters) were used. In the liquid system, the mobile phase A was 10mM ammonium formate aqueous solution (NH4COOH), pH 9.0; the mobile phase B was 90% acetonitrile aqueous solution containing 10mM ammonium formate, pH 9.0.

[0223] ① Dissolve and resuspend 4 mg of desalted peptide fragments in 0.5 mL of mobile phase A in each sample;

[0224] ② Prepare the liquid chromatography system and clean the system with at least 50 mL of fractionated mobile phase B, then equilibrate the system with at least 50 mL of fractionated mobile phase A;

[0225] ③ Dissolve 4 mg of desalted peptide in 0.5 mL of mobile phase A and resuspend the protein sample;

[0226] ④ Use gradient mobile phase to fractionate the sample. The specific gradient design is as follows:

[0227] 0.01-15 minutes, 5%-5% B;

[0228] 15-105 min, 5-40% B;

[0229] 105-108 min, 40-65% B;

[0230] 108-110 min, 65-95% B;

[0231] 110-120 min, 95-95% B.

[0232] Liquid phase flow rate: 1mL / min. Automatic collector collects at 1 tube / min. Collect about 120 tubes in total.

[0233] ⑤Drain the distillation tube after collection.

[0234] (6) Perform mass spectrometry detection and protein identification on the peptide fragments in each fractionation tube after drying:

[0235] The specific experimental process is as mentioned above, or as follows:

[0236] The collected fraction samples were resuspended in 5vt% formic acid solution to obtain about 1 μg of the sample, and then loaded onto a mass spectrometer (Thermo Q Exactive HF-X) for detection. The mass spectrometer was connected in series with a nanoliter liquid chromatography system (Thermo EASY-nLC 1200).

[0237] Liquid chromatography conditions:

[0238] Laboratory-made chromatographic column: Reprosil pure C18-AQ, inner diameter 75μm, length 25cm, filled with C18 microspheres, C18 microspheres with a particle size of 1.9μm; column temperature: 50°C; mobile phase A: contains 0.2vt% formic acid, 3vt% DMSO, and the balance is deionized water; mobile phase B: 80vt% acetonitrile, 0.2vt% formic acid, 3vt% DMSO, and the balance is deionized water; gradient elution: phase B 5-25% (0-80 minutes), phase B 25-40% (80-110 minutes), phase B 40%-65% (110-115 minutes), phase B 65%-95% B (115-120 minutes); elution time is 120 minutes; injection flow rate is 0.3μL / min.

[0239] Mass spectrometry conditions:

[0240] The mass spectrometry data were collected in DDA mode of Thermo Q Exactive_HF-X with the following parameters: spray voltage 2.1 KV; capillary temperature 320 °C; primary full scan resolution 60,000 (at 200 m / z), AGC target: 3e6, Maximum ion time: 50 ms, primary mass scan range 350-1500; Top N: 20; secondary full scan resolution 15,000 (at 200 m / z), AGC target: 1e5, Maximum ion time: 105 ms, secondary scan Fixed firstmass 100 m / z; fragmentation mode HCD, fragmentation normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s.

[0241] Peptide retrieval: The analysis of mass spectrometry raw data was processed by searching the database using Thermo Proteome Discoverer 2.5 software. The human protein sequences reviewed in the Swiss-Prot protein database were used as the database. The main search parameters were as follows: carbamidomethyl on cysteine ​​was set as a fixed modification parameter; oxidative modification on methionine and acetylation modification on the N-terminus of the protein were set as variable modification parameters. Trypsin was selected for specific enzyme cleavage, and the maximum number of missed cleavages allowed for the peptide was 2.

[0242] (7) Experimental results:

[0243] Using this ultra-deep mass spectrometry detection process, the number of proteins identified in the four samples was 2946, 2172, 3058, and 1379, respectively. A total of 4073 proteins were detected, which means that the number of plasma proteins detected can be increased from 2946 to 4073 by the method of plasma PEG precipitation and albumin filtration. The specific experimental design and process are as follows Figure 7 , the experimental results are shown in Figure 8 As can be seen from the figure, 2946 proteins can be detected in the original plasma. After the three high-abundance plasma proteins were removed, a total of 4073 proteins were finally detected. In addition, by comparing with the published major plasma protein databases or major research reports on plasma proteomes, the method of the present invention detected a total of 267 new plasma proteins, see Fig. 9 (Note: Fig. 9 The shaded area in B is the protein detected only in this sample).

[0244] The present invention provides a method and idea for a mass spectrometry pretreatment method for protein in plasma. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A method for pre-treatment of plasma protein by mass spectrometry detection, characterized in that: The steps include: (1) Polyethylene glycol precipitated plasma and albumin removal: the plasma sample is mixed with a surfactant, and then polyethylene glycol precipitated plasma is added to obtain a first mixed solution; the first mixed solution is centrifuged to obtain a precipitate A and a supernatant A; a portion of the supernatant A is centrifuged through an albumin removal centrifugal column to remove albumin, and a supernatant filtrate A is obtained; another portion of the supernatant A is taken and polyethylene glycol precipitated plasma is added to obtain a second mixed solution; the second mixed solution is centrifuged to obtain a precipitate B and a supernatant B; the supernatant B is centrifuged through an albumin removal centrifugal column to remove albumin, and a supernatant filtrate B is obtained; (2) Sample precipitation: the plasma sample is mixed with anhydrous acetonitrile for precipitation, and the precipitate C is collected; trichloroacetic acid is added to the supernatant filtrate A and the supernatant filtrate B for precipitation, respectively, and the supernatant filtrate A and the supernatant filtrate B are centrifuged respectively, and the precipitates are washed respectively, and then centrifuged again to obtain precipitates D and E respectively; (3) Proteolysis and reduction alkylation: the precipitate B, the precipitate C, the precipitate D, and the precipitate E are subjected to proteolysis treatment, protein reduction treatment, protein alkylation treatment, and quenching reaction treatment in sequence to obtain peptide samples; (4) Peptide desalting: The peptide sample obtained in step (3) is loaded onto a desalting column, and a desalting washing solution is used for desalting and washing, followed by adding a desalting eluent for elution, and the eluent containing the peptide is collected and dried to obtain the desalted peptide; (5) Reverse liquid phase chromatography fractionation under alkaline conditions: the desalted peptide fragments obtained in step (4) are subjected to reverse liquid phase chromatography fractionation, and the peptide fragment fractions obtained are collected and dried; (6) Mass spectrometry detection and protein identification: The peptide samples collected by fractionation in step (5) are injected into a liquid chromatography-mass spectrometer for mass spectrometry detection; the raw mass spectrometry data are analyzed using a protein database; In step (1), the albumin removal centrifugal column is a Cibacin blue agarose gel column; In step (1), the mixed solution obtained by mixing the plasma sample and the surfactant is mixed with a polyethylene glycol solution with a PEG6000 concentration of 18-22% g / mL to obtain a first mixed solution containing a PEG6000 concentration of 8-12% g / mL; and the supernatant A is taken from another part and the polyethylene glycol solution with a PEG6000 concentration of 30% g / mL is added to precipitate the plasma to obtain a second mixed solution containing a PEG6000 concentration of 20% g / mL.

2. The method according to claim 1, characterized in that In step (1), the plasma is human plasma or animal plasma; the surfactant is 5% to 10% Triton X-100; the volume ratio of the plasma sample to the surfactant is 9:1; and the solvent of the polyethylene glycol solution is a PBS buffer solution with a pH of 7.3 to 7.

5.

3. The method according to claim 1, characterized in that In step (2), the volume ratio of the plasma sample to anhydrous acetonitrile is 1:9; the volume ratio of the supernatant filtrate A to trichloroacetic acid is 9:1; the volume ratio of the supernatant filtrate B to trichloroacetic acid is 9:1; and the washing is performed using anhydrous acetonitrile.

4. The method according to claim 1, characterized in that: In step (3), the precipitate B, the precipitate C, the precipitate D, and the precipitate E are respectively dissolved with a urea solution containing Lys-C enzyme, and after the precipitates are dissolved, a buffer solution and trypsin are added for enzymatic hydrolysis; after the enzymatic hydrolysis is completed, dithiothreitol is added to the enzymatic hydrolyzate of each sample for reduction reaction, and after the reaction is completed, iodoacetamide is added for alkylation reaction, and after the reaction is completed, dithiothreitol is added again for quenching reaction to obtain a peptide sample.

5. The method according to claim 4, characterized in that In step (3), in the urea solution, the solute is urea, the solvent is 25-50 mM, pH=8.4~8.6 TEAB buffer, and the concentration of urea is 8 mol / L; in the urea solution containing Lys-C enzyme, the concentration of Lys-C enzyme is 0.020~0.030 μg / μL; the amount of Lys-C enzyme used, based on the plasma sample required for the initial enzymatic hydrolysis of each sample, the mass volume ratio of the Lys-C enzyme to the plasma sample is 1 μg:9 μL; the precipitate B, the precipitate C, the precipitate D, and the precipitate E are respectively dissolved with the urea solution containing Lys-C enzyme, and the buffer is added after the precipitate is dissolved. The amount of the buffer is to dilute the urea in the system to a final concentration of 2 mol / L; the mass ratio of the trypsin to the Lys-C enzyme is 1:1; the enzymolysis is carried out at room temperature for 12-24 hours; the dithiothreitol exists in the form of an aqueous solution, and dithiothreitol is mixed with water to obtain a dithiothreitol aqueous solution; in the system after the dithiothreitol aqueous solution and the enzymolysis solution are mixed, the final concentration of dithiothreitol is 2 mmol / L; the reduction reaction, the reaction temperature is room temperature, and the reaction time is 0.75-1.25 h; the iodoacetamide exists in the form of an aqueous solution, and iodoacetamide is mixed with water to obtain an iodoacetamide aqueous solution; in the system after the iodoacetamide aqueous solution and the reaction solution obtained after the reduction reaction are mixed, the final concentration of iodoacetamide is 5 mmol / L; the alkylation reaction, the reaction temperature is room temperature, and the reaction time is 0.5 h; in the system after the dithiothreitol aqueous solution and the reaction solution obtained after the alkylation reaction are mixed, the final concentration of dithiothreitol is 5 mmol / L; the quenching reaction, the reaction temperature is room temperature, and the reaction time is 15 min.

6. The method according to claim 1, characterized in that In step (4), the desalting column is a C18 desalting column; the desalting cleaning solution is a 0.1 vt% formic acid aqueous solution; the desalting eluent is a 60-65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid; The peptide desalting is carried out according to the following steps: The peptide sample obtained in step (3) is mixed with an 8-12 vt% trifluoroacetic acid aqueous solution and then loaded onto a desalting column, desalted and washed with a 0.1 vt% formic acid aqueous solution, and then eluted with a 60-65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid, and the eluate containing the peptide is collected and dried to obtain the desalted peptide; Wherein, the volume ratio of the peptide sample to the 8-12vt% trifluoroacetic acid aqueous solution is 9:

1.

7. The method according to claim 6, characterized in that The peptide sample obtained in step (3) was mixed with a 10 vt% trifluoroacetic acid aqueous solution and loaded onto a desalting column. A 0.1 vt% formic acid aqueous solution was used for desalting and washing. Subsequently, a 65 vt% acetonitrile aqueous solution containing 0.1 vt% formic acid was added for elution. The eluate containing the peptide was collected and dried to obtain the desalted peptide.

8. The method according to claim 1, characterized in that In step (5), the desalted peptide obtained in step (4) is subjected to chromatographic fractionation by reverse liquid chromatography; the desalted peptide is dissolved and resuspended in mobile phase A, and then subjected to chromatographic fractionation by a liquid chromatograph, and the peptide fractions obtained are collected and dried; The reverse liquid chromatography is performed using a liquid chromatograph, and the specific chromatographic conditions of the liquid chromatograph are as follows: Chromatographic column: C18 chromatographic column; fractionation mobile phase A: 8~12 mM ammonium formate aqueous solution, pH=9.0; fractionation mobile phase B: 90vt% acetonitrile aqueous solution containing 8~12 mM ammonium formate, pH=9.0; gradient elution, the specific gradient design is as follows: 0.01-15 minutes, 5%-5% mobile phase B; 15-105 minutes, 5-40% mobile phase B; 105-108 minutes, 40-65% mobile phase B; 108-110 minutes, 65-95% mobile phase B; 110-120 minutes, 95-95% mobile phase B; flow rate: 1 mL / min.

9. The method according to claim 1, characterized in that: In step (6), the peptide sample collected by fractionation in step (5) is resuspended in a formic acid aqueous solution and injected into a liquid chromatography-mass spectrometer for mass spectrometry detection. The specific chromatographic conditions are as follows: Liquid chromatography conditions: Chromatographic column: Reprosil pure 120Å C18-AQ, inner diameter of 75 µm, length of 25 cm, filled with C18 microspheres, particle size of C18 microspheres is 1.9 µm; column temperature: 50°C; mobile phase A: contains 0.2vt% formic acid, 3vt% DMSO, and the balance is deionized water; mobile phase B: 70~80vt% acetonitrile, 0.2vt% formic acid, 3vt% DMSO, and the balance is deionized water; gradient elution: 0~80 minutes, 5-25% mobile phase B; 80~110 minutes, 25-40% mobile phase B; 110~115 minutes, 40%-65% mobile phase B; 115~120 minutes, 65%-95% mobile phase B; elution time is 120 minutes; injection flow rate is 0.3 μL / min; Mass spectrometry conditions: The mass spectrometry data were collected in DDA mode with the following parameters: spray voltage 2.1 KV; capillary temperature 320 °C; primary full scan resolution 60,000 at 200 m / z, AGC target: 3e6, Maximum ion time: 50 ms, primary mass scan range 350-1500; Top N: 20; secondary full scan resolution 15,000 at 200 m / z, AGC target: 1e5, Maximum ion time: 105 ms, secondary scan Fixed first mass 100 m / z; fragmentation mode HCD, fragmentation normalized collision energy (NCE) 29, dynamic exclusion time 30.0 s; The analysis of mass spectrometry raw data was based on human protein sequences as the database, and the search parameters were as follows: carbamidomethylation on cysteine ​​was set as a fixed modification parameter; oxidation modification on methionine and acetylation modification on the N-terminus of the protein were set as variable modification parameters; trypsin was selected as the specific enzyme cleavage, and the maximum number of missed cleavages allowed for the polypeptide was 2.

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