CN109839303B

CN109839303B - A cross-linked peptide enrichment method and its application in the study of protein interactions

Info

Publication number: CN109839303B
Application number: CN201711226826.XA
Authority: CN
Inventors: 张丽华; 方菲; 赵群; 安雨馨; 杨开广; 张玉奎
Original assignee: Dalian Institute of Chemical Physics of CAS
Current assignee: Dalian Institute of Chemical Physics of CAS
Priority date: 2017-11-29
Filing date: 2017-11-29
Publication date: 2021-06-08
Anticipated expiration: 2037-11-29
Also published as: CN109839303A

Abstract

本发明涉及一种交联肽段富集方法及其在蛋白质相互作用研究中的应用。使用两侧具有反应活性基团且连接臂上含有邻二羟基基团的交联剂将细胞内蛋白质复合体进行交联并酶解，随后采用硼亲和材料对交联肽段进行选择性富集和高效释放。该方法具有操作简便、高效、高通量、高可信度的优点并应用于蛋白质相互作用的分析。The invention relates to a cross-linked peptide enrichment method and its application in protein interaction research. The intracellular protein complex was cross-linked and enzymatically hydrolyzed using a cross-linking agent with reactive groups on both sides and ortho-dihydroxy groups on the linker, and then the cross-linked peptides were selectively enriched with boron affinity materials. collection and efficient release. This method has the advantages of simple operation, high efficiency, high throughput and high confidence, and can be applied to the analysis of protein interactions.

Description

Crosslinked peptide enrichment method and application thereof in protein interaction research

Technical Field

The invention relates to a cross-linked peptide enrichment method and application thereof in protein interaction research.

Background

The cross-linking mass spectrometry technology is a new technology developed in more than ten years, and utilizes a chemical cross-linking agent to connect two amino acids which are close enough to each other in the intracellular space and can react with the cross-linking agent by covalent bonds, and then utilizes proteomics based on the mass spectrometry technology to analyze cross-linking products so as to realize the analysis of the spatial structure of proteins and the interaction mode between proteins (Sinz, A., Expert Rev. proteomics, 2014,11(6): 733-743.).

However, the use of conventional cross-linking mass spectrometry techniques places high demands on mass spectrometric identification. Because the actual crosslinking efficiency of each site is usually far lower than 100%, the abundance of the crosslinked peptide fragments is low, the number of crosslinking spectrograms is small, the signals are poor, and the identification of the crosslinking spectrograms is difficult. In addition, complex protein complex samples need to be cleaved by enzyme before they can be analyzed by mass spectrometry. Cleavage of the cross-linked protein at different positions results in three products: a. the single peptide modified by the cross-linking agent, namely one end of the cross-linking agent is hydrolyzed, and only one end of the cross-linking agent reacts with the peptide segment (Type-0 peptide segment); b. the peptide segment is internally crosslinked, namely the binding sites at the two ends of the crosslinking agent are on the same peptide segment (Type-1 Type crosslinking peptide segment); c. the peptide segments are cross-linked, namely the binding sites at the two ends of the cross-linking agent are respectively on the two peptide segments (Type-2 cross-linked peptide segments). Because the hydrolysis rate of the reaction groups on both sides of the crosslinking reagent is high, the content of the Type-0 peptide fragment is far higher than that of Type-1 and Type-2 crosslinking peptide fragments required by protein interaction analysis.

In order to improve the abundance of the cross-linked peptide, cross-linking reagents containing different types of enrichment groups (such as alkynyl-azide, biotin-avidin and the like) have been developed, however, the cross-linked peptide cannot be separated from the Type-0 peptide by using the cross-linking reagent containing the conventional enrichment group because all three products contain the cross-linking reagents. In addition, due to the addition of specific groups, the arm length is generally longer than that of a simple crosslinking agent, which results in a decrease in crosslinking efficiency and a weakening of the distance-limiting effect between the crosslinking sites. Meanwhile, the introduction of large molecular weight groups can cause the crosslinked peptide fragments to be more difficult to fragment in mass spectrum.

Disclosure of Invention

In order to overcome the defects that the conventional chemical crosslinking method can not realize selective enrichment of the crosslinked peptide segment, modification groups can be introduced into the crosslinked peptide segment and the like, the invention provides a method for crosslinking and carrying out enzymolysis on an intracellular protein complex by using a crosslinking agent containing an o-dihydroxy group on a connecting arm, and then selectively enriching the crosslinked peptide segment by using a boron affinity material and efficiently releasing the crosslinked peptide segment. The method has the advantages of simple and convenient operation, high efficiency, high flux and high reliability, and is applied to the structural analysis of the protein and the analysis of the protein complex interaction binding site.

In order to achieve the purpose, the invention adopts the technical scheme that:

(1) preparing a protein/cell sample solution and a cross-linking agent solution: for live cell samples obtained by trypsinization or cell scraping digestion, washing with one or more of ammonium bicarbonate buffer salt solution, phosphate buffer salt solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffer salt solution or tris (hydroxymethyl) aminomethane buffer salt solution with pH of 7.1-10, and buffer solution containing no group reactive with the reactive group on the crosslinking agent used, to remove the culture solution and suspend the cells; for a single protein sample or a mixed protein sample, preparing a protein solution with the concentration of 1 mu g/mL-100mg/mL by using water or buffer solution; preparing a cross-linking agent solution with the concentration of 1 mu M-1M by using water, buffer solution or one or more than two organic solvents of acetonitrile, organic alcohols, organic acids, DMF or DMSO, wherein the two sides of the cross-linking agent contain one or two reaction groups of succinimide, halogenated aromatic hydrocarbon, imidic acid ester, maleimide, 2-mercaptopyridine, thiosulfonate, halogenated acetyl, carbodiimide, isocyanate, hydrazide, phenyl azide, diazirine and the like, and the connecting arm contains an ortho-dihydroxy group;

(2) and (3) crosslinking reaction: adding the cross-linking agent solution into a cell sample or a protein sample for reaction, wherein the cell concentration in the reaction system is 10 for the cell sample⁶-10⁹The concentration of the protein in the reaction system is 1nM to 1mM and the concentration of the cross-linking agent is 10nM to 100mM for the protein sample; the reaction condition is that the reaction is carried out for 10min-2h at 15-40 ℃ or 10min-10h at 0-10 ℃;

(3) removal of excess crosslinker: centrifuging a cell sample to remove a reaction solution in the cell sample, adding a lysis solution into cells, and performing cell lysis by using one or two cell lysis methods of a mechanical lysis method or a high-temperature incubation method to obtain a crosslinked protein sample; adding a reaction stop solution into a reaction system for a protein sample, or removing the reaction solution through dialysis, a filter membrane or gel to obtain a cross-linked protein sample, and reacting for 10min-2h at room temperature or for 10min-10h on ice;

(4) solubilization, denaturation and reduction of protein samples: dissolving protein sample with a solution prepared from acidic buffer solution (pH 1-6.5) such as formic acid, trifluoroacetic acid, trichloroacetic acid or acetic acid, etc. or alkaline buffer solution (pH 7.5-14) dissolved with surfactant or organic solvent, adding one or more of reducing agents such as DTT, TCEP or beta-mercaptoethanol, etc., incubating in water bath at 40-100 deg.C for 1min-10h, and simultaneously performing denaturation and reduction of protein sample;

(5) alkylation and enzymatic hydrolysis of protein samples: adding one or two of iodoacetic acid or iodoacetamide to carry out alkylation reaction on the protein sample, and adding one or more of trypsin, proteinase K, pepsin, elastase, carboxypeptidase, chymotrypsin, intracellular protease Lys-C/N and endoprotease Glu-C/N, Asp-C/N into the protein sample, wherein when more than two are used, the two are used simultaneously or sequentially;

(6) adding an enrichment material prepared from organic/inorganic materials such as agarose gel spheres, silicon spheres and polymer spheres with one or more than two boron affinity groups of monomers such as 2-carboxyphenylboronic acid, 5-carboxy-2-hydroxymethylphenylboronic acid, 4-formylphenylboronic acid, aminophenylboronic acid or 5-amino-2-hydroxymethylphenylboronic acid into the product obtained in the step (5), and reacting with a sample;

(7) washing off the peptide segment nonspecifically adsorbed on the enrichment material by using an eluent such as a salt solution or an organic solvent;

(8) releasing the peptide segment bonded on the enrichment material by using an acidic solution, desalting, freeze-drying and re-dissolving for mass spectrometry and data retrieval.

(9) The sample pretreatment method is used for researching protein interaction in the fields of organism signal path and protein structure analysis.

The invention has the following advantages:

1. the operation is simple. The experimental steps are simple, and the enrichment and release rates of the cross-linked peptide segment are high.

2. The enrichment selectivity is high. The boron affinity material has high-efficiency enrichment selectivity on the cross-linked peptide segment containing the o-dihydroxy group.

3. The recovery rate is high. The solution system is replaced by acidic solution, and the cross-linked peptide segment can be efficiently released from the boron affinity enrichment material.

4. The reliability is high. The interference of the non-crosslinked peptide segment is greatly reduced, the abundance of the crosslinked peptide segment is greatly improved, and the identification sensitivity of the crosslinked peptide segment is greatly improved, so that the reliability of the identification result is improved.

Detailed Description

Example 1

1. Cross-linking reactions of protein samples

Mu.g of Bovine Serum Albumin (BSA) sample was dissolved using 20mM 4-hydroxyethylpiperazine ethanesulfonic acid buffer salt solution (HEPES) having a pH of 7.4, the final protein concentration was 1mg/mL, bis-succinimidyl tartrate (DST) was prepared at a concentration of 25mM using dimethyl sulfoxide (DMSO), and the crosslinker was added to the BSA solution so that the final concentration was 1mM, and the reaction was carried out at room temperature for 1 hour.

2. Removal of excess crosslinker

To the reaction solution in step 1, ammonium bicarbonate solution (ABC) was added to a final concentration of 50mM to terminate the crosslinking reaction.

3. Solubilization, denaturation and reduction of protein samples

Adding urea and DTT into the BSA solution after crosslinking in the step 2 to ensure that the final concentration of the urea in the solution is 8M and the final concentration of the DTT is 10mM, and carrying out water bath reaction at 37 ℃ for 30 min.

4. Alkylation and enzymolysis of protein solutions

Iodoacetic acid was added to the BSA solution in step 3 to give a final concentration of 20mM, and the mixture was left to react for 30min under dark conditions. After the alkylation reaction was completed, the sample solution was diluted 4-fold with water, and 1. mu.g of serine protease Lys-C was added thereto, followed by reaction in a water bath at 37 ℃ for 4 hours. After the reaction, 2. mu.g of trypsin was added to the sample solution, and the reaction was carried out overnight in a water bath at 37 ℃. Desalting the enzymolysis product and freeze-drying.

5. Enrichment of Cross-Linked peptide fragments

The enzymatic product was redissolved using 50mM ammonium bicarbonate solution (ABC, pH 10) and added to silica spheres of phenylboronic acid and reacted at room temperature for 2 h.

6. Removal of non-specifically adsorbed peptide fragments

The non-specifically adsorbed peptide fragments on the silica spheres were washed with 50mM ABC solution (pH 10).

7. Release of peptide fragments

Eluent (acetonitrile: water: trifluoroacetic acid ═ 50:49:1) was added, and the reaction was carried out at room temperature for 1 hour.

8. Determination of the crosslinking sites

And (3) freeze-drying the released cross-linked peptide fragment, re-dissolving the cross-linked peptide fragment in 0.1% formic acid solution, and performing mass spectrometry.

Identification results

VTKCCTESLVNR(3)-CASIQKFGER(6)
	LCVLHEKTPVSEK(7)-CASIQKFGER(6)
LAKEYEATLEECCAK(3)-VTKCCTESLVNR(3)
	KVPQVSTPTLVEVSR(1)-HKPKATEEQLK(4)
LAKEYEATLEECCAK(3)-CASIQKFGER(6)
	CCTKPESER(4)-SLGKVGTR(4)
VHKECCHGDLLECADDR(3)-ALKAWSVAR(3)
	FKDLGEEHFK(2)-DTHKSEIAHR(4)
CASIQKFGER(6)-SLGKVGTR(4)
	RHPYFYAPELLYYANKYNGVFQECCQAEDK(16)-GACLLPKIETMREK(7)
KVPQVSTPTLVEVSR(1)-CASIQKFGERALK(6)
	DTHKSEIAHR(4)-LVTDLTKVHK(7)
TCVADESHAGCEKSLHTLFGDELCK(13)-RDTHKSEIAHR(5)
	LVTDLTKVHK(7)-ALKAWSVAR(3)
LKECCDKPLLEK(2)-FPKAEFVEVTK(3)
	YICDNQDTISSKLK(12)-FPKAEFVEVTK(3)
LAKEYEATLEECCAK(3)-ALKAWSVAR(3)
	CASIQKFGER(6)-ALKAWSVAR(3)
NYQEAKDAFLGSFLYEYSR(6)-ALKAWSVAR(3)
	DTHKSEIAHR(4)-SLGKVGTR(4)
ATEEQLKTVMENFVAFVDK(7)-KVPQVSTPTLVEVSR(1)
	CASIQKFGER(6)-LSQKFPK(4)
CCTKPESER(4)-LSQKFPK(4)
	SHCIAEVEKDAIPENLPPLTADFAEDK(9)-CCTKPESERMPCTEDYLSLILNR(4)
CCTKPESERMPCTEDYLSLILNR(4)-GACLLPKIETMR(7)
	SHCIAEVEKDAIPENLPPLTADFAEDK(9)-YICDNQDTISSKLK(12)
RHPYFYAPELLYYANKYNGVFQECCQAEDK(16)-ECCDKPLLEK(5)
	ECCKDPLLEKSHCIAEVEK(10)-ADLAKYICKNQDTISSK(5)

Example 2

Crosslinking reaction of BSA protein sample; removing the redundant cross-linking agent; the steps of dissolving, denaturation and reduction of the protein sample, alkylation, enzymolysis, enrichment of cross-linked peptide fragments, removal of non-specific adsorption peptide fragments and release of the peptide fragments are the same as in example 1.

2. Fractionation of enriched cross-linked peptide fragments and determination of cross-linking sites

Desalting the cross-linked peptide segment released in the step (1), freeze-drying, classifying a sample by using cation exchange separation, desalting, freeze-drying and re-dissolving by using 0.1% formic acid solution for mass spectrometry.

Identification results

Example 3

1. Cross-linking reactions of protein samples

Mu.g of a rabbit creatine kinase protein sample (CK) was dissolved using 50mM Phosphate Buffered Saline (PBS) at pH 7.4 to a final protein concentration of 1mg/mL, 25mM DST was prepared using Dimethylformamide (DMF), and a crosslinking agent was added to the CK solution to a final concentration of 1mM and reacted at room temperature for 1 hour.

2. Removal of excess crosslinker

Tris buffer (Tris) was added to the reaction mixture in step 1 to a final concentration of 50mM to terminate the crosslinking reaction.

3. Solubilization, denaturation and reduction of protein samples

Adding urea and DTT into the CK solution after crosslinking in the step 2 to ensure that the final concentration of the urea in the solution is 8M and the final concentration of the DTT is 10mM, and carrying out water bath reaction at 37 ℃ for 30 min.

4. Alkylation and enzymolysis of protein solutions

Iodoacetic acid was added to the CK solution in step 3 to a final concentration of 20mM, and the mixture was reacted for 30min with exclusion of light. After the alkylation reaction was completed, the sample solution was diluted 4-fold with water, and 1. mu.g of serine protease Lys-C was added thereto, followed by reaction in a water bath at 37 ℃ for 4 hours. After the reaction, 2. mu.g of trypsin was added to the sample solution, and the reaction was carried out overnight in a water bath at 37 ℃. Desalting the enzymolysis product and freeze-drying.

5. Enrichment of Cross-Linked peptide fragments

The enzymatic product was redissolved using 50mM ammonium bicarbonate solution (ABC, pH 7) and added to a magnetic sphere of phenylboronic acid and reacted at room temperature for 2 h.

6. Removal of non-specifically adsorbed peptide fragments

The non-specifically adsorbed peptide fragments on the magnetic spheres were washed with 50mM ABC solution (pH 7).

7. Release of peptide fragments

8. Determination of the crosslinking sites

Identification results

SIKGYTLPPHCSR(3)-IEEIFKK(6)
	VISMEK(3)-FCVGLQKIEEIFKK(7)
VISEKGGNK(5)-GGVHVKLAHLSK(6)
	LAHLSKHPK(6)-LQKR(3)
TGKSIKGYTLPPHCSRGER(3)-LSVEALNSLTGEFKGK(2)
	LNYKSEEEYPDLSK(4)-AVEKLSVEALNSLTGEFK(4)
GKYYPLK(2)-VISMEKGGNMKEVFRR(6)
	LNYKSEEEYPDLSK(4)-VLTPDLYKK(8)
GGVHVKLAHLSK(6)-LQKR(3)
	GGDDLDPHYVLSSR(9)-VISEKGGNK(5)
GGVHVKLAHLSK(6)-LEKGQSIDDIPAQK(3)

According to the invention, a cross-linking agent containing an o-dihydroxy group on a connecting arm is used for cross-linking cells or an extracted protein sample, after the protein sample is subjected to enzymolysis, a boron affinity material is adopted for selectively enriching inter-peptide cross-linked peptide fragments, and since carboxyl in a Type-0 peptide fragment can prevent o-dihydroxy on the cross-linking agent from acting with boron atoms under an alkaline condition, high selective enrichment of Type-1 and Type-2 cross-linked peptide fragments can be realized. Meanwhile, the solution system is replaced by an acid environment, the cross-linked peptide segment can be efficiently released from the boron affinity material, and a modifying group is not introduced. And (3) subjecting the obtained cross-linked peptide segment to mass spectrometry so as to obtain the protein structure and protein interaction information. The method has the advantages of simple and convenient operation, high efficiency, high flux and high reliability, and can be applied to the structural analysis of protein and the analysis of the protein complex interaction binding site.

Claims

1. A method for enrichment of cross-linked peptide segments, characterized in that: using a cross-linking agent containing an adjacent dihydroxy group on the connecting arm to cross-link cells or the extracted protein sample, the cross-linked protein sample is subjected to cross-linking. After enzymatic hydrolysis, the cross-linked peptides are selectively enriched with boron affinity materials to obtain protein structure and protein interaction information;

Specifically include:

(1) For cell samples, wash with buffer to remove the culture medium and suspend the cells; for extracted protein samples, use water or buffer to prepare a solution; for cross-linking agents, use water, buffer or organic solvent to prepare into a solution;

(2) adding the cross-linking agent solution to the cell sample solution or the protein sample solution for reaction;

(3) For cell samples, the reaction solution in the reaction system is discarded by centrifugation, and then the cell samples are lysed to obtain cross-linked protein samples; The gel removes the reaction solution to obtain a cross-linked protein sample;

(4) Dissolve the cross-linked protein sample with an acidic buffer solution with a pH of 1-6.5 or an alkaline buffer solution with a pH of 7.5-14 to dissolve the cross-linked protein sample, add a reducing agent, and incubate at high temperature , denaturation and reduction of protein samples at the same time;

(5) After adding an alkylating reagent to perform an alkylation reaction on the denatured and reduced protein sample, add a protease solution to the protein sample for enzymatic hydrolysis, and desalt the enzymatic hydrolysis product and freeze-dry it;

(6) adding enrichment material with boron affinity group to the product of step (5);

(7) Use the eluent to wash off the non-specifically adsorbed peptides on the enrichment material;

(8) Use an acidic solution to release the peptides bound to the enriched material, desalinate, freeze-dry and redissolve, perform mass spectrometry analysis and data retrieval;

The concentration of the crosslinker described in step (1) is 1 µM-1 M;

The cross-linking agent is a chemical compound with active groups reacting with amino groups on the protein on both sides, and the active groups are one of succinimide groups, halogenated aromatic hydrocarbon groups, and imidate groups. Or two kinds; or a chemical compound with active groups reacting with sulfhydryl groups on the protein on both sides, and the active groups are maleimide groups, 2-mercaptopyridine groups, thiosulfonic acid groups, halogenated groups One or two of the acetyl groups; or a chemical compound with active groups reacting with carboxyl groups on the protein on both sides, and the active groups are one of carbodiimide groups, isocyanate groups or Two; or a chemical compound with active groups on both sides that react with sugar chains on the protein, and the active group is one or both of hydrazide groups and amino groups; The chemical substance of the active group of any group reaction, the active group is one or two of the phenyl azide group and the diaziridine group; the crosslinking agent, the active reactive groups on both sides thereof The groups are respectively any of the above-mentioned reactive groups; the cross-linking agent, the chemical enrichment group on its connecting arm, is an adjacent dihydroxy group;

The reaction termination liquid described in step (3) is a substance having a group capable of reacting with the reactive groups on both sides of the crosslinking agent;

The monomer of the boron affinity group described in step (6) is 2-carboxyphenylboronic acid, 5-carboxy-2-hydroxymethylphenylboronic acid, 4-formylphenylboronic acid, aminophenylboronic acid or 5-amino- One or more of 2-hydroxymethylphenylboronic acid;

The matrix of the enriched material is agarose gel spheres, silicon spheres or polymer spheres organic/inorganic materials;

The eluent described in step (7) is one of the following: sodium chloride solution with a concentration of 0.1-8 M, potassium chloride solution of 0.1-8 M, sodium carbonate solution of 0.1-1 M, 2-10 M in urea, 1-8 M in guanidine hydrochloride, or 10-1000 mM in ammonium bicarbonate; acetonitrile, methanol, isopropanol; sodium dodecyl sulfonate, Triton X-100, Chaps, Tween;

The cross-linked peptide release solution described in step (8) is an acidic solution with a pH of 1-6.5, and the acidic solution is one or more of formic acid, trifluoroacetic acid, trichloroacetic acid or acetic acid solution.

2. The method according to claim 1, characterized in that: the buffer described in step (1) is ammonium bicarbonate buffered saline solution, phosphate buffered saline solution, 4-hydroxyethylpiperidine with pH of 7.1-10 One or more of oxazine ethanesulfonic acid buffered saline solution or tris buffered saline solution, and does not contain a group that can react with the reactive group on the used crosslinking agent;

The cell sample described in step (1) is a live cell sample obtained by trypsin digestion or cell scraping digestion; the protein sample described in step (1) is a single protein sample or a mixed protein sample of two or more proteins, The volume ratio of the prepared protein to the solution is 1 µg/mL-100 mg/mL; the organic solvent described in step (1) is acetonitrile, organic alcohols, organic acids, dimethylformamide (DMF) Or one or more of dimethyl sulfoxide (DMSO).

3. The method according to claim 1, wherein in the reaction system described in step (2), the cell concentration is 10 ⁶ -10 ⁹ cells/mL, the final protein concentration is 1 nM-1 mM, and the cross-linked The final concentration of the reagent is 10 nM-100 mM; the reaction conditions are 15-40°C for 10 min-2 h or 0-10°C for 10 min-10 h.

4 . The pretreatment method according to claim 1 , wherein the reaction conditions for protein samples are 15-40° C. for 10 min-2 h, and the reaction conditions for cell samples are 0-10° C. for 10 min-10 h. 5 . .

5. The pretreatment method according to claim 1, wherein the cell lysis method in step (3) is one or both of mechanical lysis or high temperature incubation;

The above-mentioned mechanical lysis method, specifically, after adding a protease inhibitor to the mixture of the protein extract and the biological sample, using a homogenizer, a sonicator, a mortar or a tissue masher to lyse the protein sample or the cell sample;

The above-mentioned protease inhibitor is one or more of the following substances: 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, aprotinin, aprotinin, sodium pyrophosphate, trans-epoxy One or two of succinyl-L-leucinamido(4-guanidino)butane, disodium EDTA, leupeptin or pepstatin A, phenylmethanesulfonyl fluoride more than one species;

The concentration range of each protease inhibitor in the protein extract is between 1-200 mg/mL;

The above-mentioned high-temperature incubation method specifically involves incubating the mixture of the protein extract and the biological sample in a water bath at 40-100 °C for 1-60 min.

6. The method according to claim 1, characterized in that: in the protein sample dissolving solution described in step (4), the acidic buffer solution with pH of 1-6.5 is formic acid, trifluoroacetic acid, trichloroacetic acid or Acetic acid solution; the alkaline buffered buffer solution whose pH is 7.5-14 is ammonium bicarbonate buffered saline solution, phosphate buffered saline solution, 4-hydroxyethylpiperazine ethanesulfonic acid buffered saline solution or tris(hydroxymethyl)aminomethane One or more of buffered saline solutions;

In the protein dissolving solution, the surfactant includes:

1) Sodium dodecyl sulfonate, sodium deoxycholate, alkyl glycosides, 100 (polyethylene glycol octyl phenyl ether) (Triton X-100), 3-[3-(cholamidopropyl) two One or more of methylamino] propanesulfonic acid inner salt (CHAPS), RapiGest SF or ethyl phenyl polyethylene glycol (NP-40), the mass volume concentration is based on the quality of the surfactant (g is unit) to the volume of the buffer solution (in mL) as a ratio of 0.1% to 30%;

2) The cationic part is one or more of imidazoles, pyridines, quaternary ammoniums, or quaternary phosphonium cations whose alkyl chain part contains more than 2 carbons; the anionic part is halogen ion, NO ₃ ^- , ClO Ionic liquid of one or more of ₄ ^- , AlCl ₄ ^- , BF ₄ ^- , PF ₄ ^- , CF ₃ COO ^- , CF ₃ SO ₃ ^- , (CF ₃ SO ₂ ) ₂ N ^- or SbF ₆ ^- ; The mass volume concentration is 0.1%-30% according to the ratio of the mass of the ionic liquid (in g) to the volume of the alkaline buffer solution (in mL);

3) One or more of urea, thiourea or guanidine hydrochloride, and the molar concentration is 0.1-20 M according to the ratio of the amount of detergent to the volume of the buffer solution;

The organic solvent is an organic alcohol or an organic acid, and the volume concentration is 0.1%-100% according to the volume ratio of the organic solvent and the buffer solution;

The reducing agent is one or more of dithiothreitol (DTT), tris(2-carboxyethyl) phosphine (TCEP) or β-mercaptoethanol, and the molar concentration is based on the amount of the reducing agent. The ratio to the volume of alkaline buffer solution is calculated as 0.1-1000 mM;

The high temperature incubation is specifically incubating the mixture of the protein extract and the biological sample in a water bath at 40-100° C. for 1 min-10 h.

7. The method according to claim 1, wherein the alkylating reagent described in step (5) is one or both of iodoacetic acid or iodoacetamide, dissolved in the above-mentioned alkaline buffer solution The molarity of the latter is 1-200 mM;

Described protease is trypsin, proteinase K, pepsin, elastase, carboxypeptidase, chymotrypsin, intracellular protease lysine-C/N, endoproteinase Glu-C/N, Asp-C One or more than two kinds of /N, when more than two kinds of enzymes are used, the selected enzymes can be used simultaneously or sequentially, and the mass ratio of protease to protein is 1:500-500:1.

8. The method according to claim 1, which is used for the application of protein interaction research in the field of body signaling pathway and protein structure analysis.