阅读说明：本技术 基于特异性交联酪氨酸构建蛋白质三维结构的方法 (Method for constructing three-dimensional structure of protein based on specific cross-linked tyrosine ) 是由 魏忠林 崔利利 马咏歌 郑连友 于 2020-05-15 设计创作，主要内容包括：本发明的基于特异性交联酪氨酸构建蛋白质三维结构的方法,属于交联质谱技术领域。具体包括化学交联反应、酶解、电化学还原、质谱分析、重建蛋白质三维信息等步骤。本发明将近期兴起的电点击化学技术与电化学、高分辨质谱等多种先进分析测量手段综合运用,进行蛋白质空间结构的研究,并通过首次设计合成新型特异性靶向酪氨酸的交联剂和EC-EC/LC-MS<Sup>n</Sup>实验方法补充交联-质谱现有的技术,为蛋白质参与的高度复杂和动态的相互作用网络等研究提供理论依据和实验基础。(The invention discloses a method for constructing a three-dimensional structure of protein based on specific cross-linked tyrosine, belonging to the technical field of cross-linked mass spectrometry. The method specifically comprises the steps of chemical crosslinking reaction, enzymolysis, electrochemical reduction, mass spectrometry, reconstruction of protein three-dimensional information and the like. The invention comprehensively uses the recently-developed electro-click chemistry technology and various advanced analysis and measurement means such as electrochemistry, high-resolution mass spectrometry and the like to research the space structure of protein, and synthesizes a novel cross-linking agent of specific target tyrosine and EC-EC/LC-MS by first design n The experimental method supplements the existing technology of cross-linking-mass spectrometry, and provides theoretical basis and experimental basis for researches such as highly complex and dynamic interaction networks involved by protein.)

1. A method for constructing a three-dimensional structure of protein based on specific cross-linked tyrosine comprises the following specific steps:

(1) chemical crosslinking reaction: dissolving a protein sample in 100mM PB buffer solution with the pH value of 7.40 to obtain 1mM sample solution, reacting the sample solution and a cross-linking agent at a molar ratio of 1: 2-30 at the voltage of 0.36V at room temperature for 4h, wherein a three-electrode system is used for reaction: a graphite working electrode, a platinum counter electrode and a saturated calomel reference electrode; the protein sample is angiotensin II, insulin or recombinant human growth hormone; the structural formula of the cross-linking agent is as follows:

(2) enzymolysis: when the protein sample is insulin or recombinant human growth hormone, performing enzymolysis reaction on a product of the crosslinking reaction in the step (1), when the protein sample is insulin, performing enzymolysis reaction by using pepsin dissolved in 1% acetic acid solution, and incubating at 37 ℃ for 6.5h, wherein the mass ratio of the pepsin to the protein is 50: 1; when the protein sample is recombinant human growth hormone, performing enzymolysis reaction by using trypsin dissolved in Tris-HCl buffer solution with the pH of 7.50 and 50mM, and incubating at 37 ℃ for 4h, wherein the mass ratio of the trypsin to the protein is 12: 1;

(3) electrochemical reduction: passing the protein sample treated in step (1) or step (2) into an electrochemical cell for electrochemical reduction using a syringe pump, applying a voltage of-3V across the electrochemical cell to cleave disulfide bonds and cross-linkers in the cross-linked protein, then treating the sample with 100mM N-ethylmaleimide solution to a final cysteine/alkylate molar ratio of 1:50, and incubating at room temperature for 2 h; the electrochemical reduction is carried out in a thin-layer electrochemical cell consisting of a lead working electrode, an Ag/AgCl reference electrode and a stainless steel auxiliary electrode, and the volume of the electrochemical cell is 7 mu L;

(4) mass spectrometry analysis: respectively analyzing the sample solution before and after electrochemical reduction reaction by using a liquid chromatography-mass spectrometer, performing liquid phase separation by using a C18 reversed phase column before mass spectrum analysis, and performing MS²The spectrogram is generated by collision induced dissociation, the energy is 15eV, and the mass spectrum acquisition mode is a primary mass spectrum and a secondary mass spectrum;

(5) reconstructing three-dimensional information of the protein: analyzing and sorting the obtained mass spectrum data, and calculating the spacing length of the cross-linking agent and the contribution distance of the tyrosine side chain by using Gaussian View 6 software; and calculating the Euclidean distance between C alpha and C alpha of the insulin and the R-hGH by utilizing PyMOL 2.3 software to obtain the three-dimensional structure information of the protein.

Technical Field

The invention belongs to the technical field of cross-linked mass spectrometry, and particularly relates to a method for constructing a three-dimensional structure of protein based on specific cross-linked tyrosine.

Background

Significant advances in Cross-linking mass spectrometry (XL-MS) have greatly driven the development of human pathology, diagnostics and therapeutics in exploring the three-dimensional structure of proteins and Protein complexes and Protein-Protein interactions (PPIs) (Nature,2016,537,347; Analytical chemistry,2017,90, 144; CN 105651852A). The presence of an enzymatic cleavage step in cross-linking mass spectrometry allows mass spectrometry to be performed at the peptide level, compared to conventional nmr spectroscopy and X-ray crystallography, and therefore the quality of the protein assembly itself is not limited (angelside Chemie international edition,2018,57, 6390). Furthermore, cross-linking mass spectrometry can be applied to membrane proteins, highly flexible protein complexes and very large protein assemblies, and is therefore also referred to as the "molecular machine" (Trends in biochemical sciences,2016,41, 20). In a conventional cross-linking mass spectrometry experimental method, a protein is firstly covalently bound with a cross-linking reagent, then is digested into a cross-linking polypeptide fragment by enzymolysis, and finally is separated and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) (2014, 11, 733; The EMBO journal,2016,35, 2634). Since a crosslinker of defined length imposes a limit on the distance between two attached residues, structural information of proteins and protein complexes can be deduced both in vitro (Science,2012,337,1348) and in vivo (Journal of Biological Chemistry,2017,292,16310).

However, the crosslinking mass spectrometry technique also faces the challenges of complex crosslinking fragments and uneven structure of the crosslinked product (Molecular)&Cellular Proteomics,2016,15,1105；Molecular&Cellular Proteomics,2010,9, 1634). Therefore, the development of various types of cleavable crosslinkers to facilitate and simplify mass spectrometric identification of crosslinked peptides has become elusive. Among these, mass-cleavable crosslinkers are favored by a large number of researchers because of their ability to obtain characteristic fragments during mass spectrometry (Analytical chemistry,2014,86, 2099; Analytical chemistry,2008,80, 9279). The Huang group of subjects successfully developed a series of novel sulfoxide-containing, mass-cleavable crosslinkers, such as: disuccinimidyl sulfoxide (DSSO) for specific cross-linking of lysine residues (Molecular&CellularProteomics,2011,10, M110.002212), dihydrazide sulfoxides specifically crosslinking carboxylic acid residues(DHSO) (Analytical chemistry,2016,88,8315), and bismaleimide sulfoxide (BMSO) (Analytical chemistry,2018,90,7600) that specifically crosslinks cysteine residues. Meanwhile, patent CN109897078A and patent CN109900814A report a preparation and analysis method of glycosidic bond mass spectrum fragmentation type cross-linking agent. These mass-labile C-S bonds selectively preferentially cleave during collision-induced dissociation, thereby generating predictable signature fragments for subsequent sequencing. To date, The main specific crosslinkers have been studied with The aim of lysine (Journal of proteomics, 2013,12, 1569; Journal of The American Society for Mass Spectrometry,2017,28,2039), glutamic acid, aspartic acid (Analytical chemistry,2018,90, 1195; European Journal of Mass Spectrometry,2008,14, 355; Proceedings of The National academy of Sciences,2014,111,9455), and cysteine, whereas less research has been done on The remaining 16 amino acids. Tyrosine (Y) is one of the essential amino acids and is widely present in many peptides and proteins, such as myoglobin, recombinant human growth hormone (r-hGH) and tyrosine protein kinase. At the same time, aromatic amino acid residues on the protein surface have the advantage of being able to produce a high degree of bioconjugation without changing the overall charge state or redox sensitivity (Journal of the American chemical Society,2004,126,15942). However, the redox potentials of these aromatic amino acid residues are similar, C (sp)²) Functionalization of-H is difficult, so there are few technical reports on the crosslinking of tyrosine residues (Journal of the American Chemical Society,2016,138,15118; journal of the American chemical society,2017,139,7152).

Disclosure of Invention

In view of the problems of the background art, the present invention is directed to a method for constructing a three-dimensional structure of a protein based on specifically crosslinked tyrosine, which can provide more secondary fragmentation information by performing electrochemical reduction of disulfide bonds without using any chemical reducing agent when analyzing disulfide bond-containing polypeptides and proteins.

The invention uses a novel cross-linking agent and protein through' electro-click chemistryCarrying out chemical crosslinking reaction, carrying out mass spectrum identification on the crosslinked proteolysis product, and leading S-S bonds in the crosslinked product to be subjected to preferential fragmentation in an electrochemical reduction mode to generate characteristic fragments. Mass relationships of these characteristic fragments and MS of the Cross-Linked peptide fragments²The fragmentation mass spectrogram can accurately determine the crosslinking sites in the protein and reduce the database retrieval scale of the protein crosslinking sites.

The specific technical scheme of the invention is as follows:

a method for constructing a three-dimensional structure of protein based on specific cross-linked tyrosine comprises the following specific steps:

(1) chemical crosslinking reaction: dissolving a protein sample in 100mM PB buffer solution with the pH value of 7.40 to obtain 1mM sample solution, reacting the sample solution and a cross-linking agent at a molar ratio of 1: 2-30 at the voltage of 0.36V at room temperature for 4h, wherein a three-electrode system is used for reaction: a graphite working electrode, a platinum counter electrode and a saturated calomel reference electrode; the protein sample is angiotensin II, insulin or recombinant human growth hormone (r-hGH); the structural formula of the cross-linking agent is as follows:

(2) enzymolysis: when the protein sample is insulin or recombinant human growth hormone, performing enzymolysis reaction on a product of the crosslinking reaction in the step (1), when the protein sample is insulin, performing enzymolysis reaction by using pepsin dissolved in 1% acetic acid solution, and incubating at 37 ℃ for 6.5h, wherein the mass ratio of the pepsin to the protein is 50: 1; when the protein sample is recombinant human growth hormone, performing enzymolysis reaction by using trypsin dissolved in Tris-HCl buffer solution with the pH of 7.50 and 50mM, and incubating at 37 ℃ for 4h, wherein the mass ratio of the trypsin to the protein is 12: 1;

(3) electrochemical reduction: passing the protein sample treated in step (1) or step (2) into an electrochemical cell for electrochemical reduction using a syringe pump, applying a voltage of-3V across the electrochemical cell to cleave disulfide bonds and cross-linkers in the cross-linked protein, then treating the sample with 100mM N-ethylmaleimide (NEM) solution to a final cysteine/alkylate molar ratio of 1:50, and incubating at room temperature for 2 h; the electrochemical reduction is carried out in a thin-layer electrochemical cell consisting of a lead Working Electrode (WE), an Ag/AgCl (3 mol/lnaacl) Reference Electrode (RE) and a stainless steel Auxiliary Electrode (AE), said electrochemical cell having a volume of 7 μ L;

(4) mass spectrometry analysis: respectively using a liquid chromatography-mass spectrometry combination instrument (LC-MS) to sample solutions before and after the electrochemical reduction reactionⁿ) The analysis was carried out by liquid phase separation, MS, using a C18 reverse phase column before mass spectrometry²The spectrogram is generated by Collision Induced Dissociation (CID), the energy is 15eV, and the mass spectrum acquisition mode is a primary mass spectrum and a secondary mass spectrum;

(5) reconstructing three-dimensional information of the protein: analyzing and sorting the obtained mass spectrum data, and calculating the spacing length of the cross-linking agent and the contribution distance of the tyrosine side chain by using Gaussian View 6 software; and calculating the Euclidean distance between C alpha and C alpha of the insulin and the R-hGH by utilizing PyMOL 2.3 software to obtain the three-dimensional structure information of the protein.

The invention comprehensively uses the recently-developed electro-click chemistry technology and various advanced analysis and measurement means such as electrochemistry, high-resolution mass spectrometry and the like to research the space structure of protein, and synthesizes a novel cross-linking agent of specific target tyrosine and EC-EC/LC-MS by first designⁿThe experimental method supplements the existing technology of cross-linking-mass spectrometry, and provides theoretical basis and experimental basis for researches such as highly complex and dynamic interaction networks involved by protein.

The invention has the following beneficial effects:

1. the tyrosine can be specifically crosslinked by adopting the electro-click chemistry technology, so that the types of crosslinking agents in the existing crosslinking-mass spectrometry research method are supplemented, and the generation of the first generation of the crosslinking agent for specifically crosslinking the tyrosine is marked.

2. By utilizing the characteristic of electrochemical cracking of disulfide bonds, a cross-linking mass spectrum identification strategy capable of generating characteristic ions is developed, and theoretical basis and experimental basis are provided for researches such as highly complex and dynamic interaction networks involved by proteins.

3. When the polypeptide and the protein containing the disulfide bonds are analyzed, the electrochemical reduction of the disulfide bonds can be realized without using any chemical reducing agent, so that more secondary fragmentation information is provided.

Drawings

Fig. 1 is based on EC cleavable crosslinkers specifically targeting tyrosine, proposing a workflow for performing crosslinking and electrochemical reduction to identify crosslinked peptides.

FIG. 2 shows MS before and after cross-linking of angiotensin II²And (4) mass spectrum.

FIG. 3 is a graph of the identified mass spectral data of insulin and cross-linking sites

FIG. 4 shows the three-dimensional structure and cross-linking sites of recombinant human growth hormone

Detailed Description