CN117946081A - Mass spectrum cleavable abnormal-shaped difunctional crosslinking agent and preparation method and application thereof - Google Patents

Abstract

The invention discloses a mass spectrum cleavable abnormal-shaped bifunctional cross-linking agent, and a preparation method and application thereof, and belongs to the field of protein structure analysis. The method is characterized by comprising two different reaction groups, namely a urea azole group and an N-hydroxysuccinimide group, and a symmetrical mass spectrum cleavable C-S bond serving as a framework structure, wherein the urea azole group is selectively and specifically crosslinked with tyrosine under the electro-click chemistry condition and specifically crosslinked with lysine under the physiological condition, the difunctional crosslinking agent is used for carrying out chemical crosslinking reaction with protein, and a mass spectrum identification is carried out on a crosslinked proteolytic product to preliminarily judge a modified peptide in the protein. The invention has the advantages of simple synthesis steps, easily obtained raw materials, low price, environmental protection and the like.

Description

Mass spectrum cleavable abnormal-shaped difunctional crosslinking agent and preparation method and application thereof

Technical Field

The invention belongs to the field of protein structure analysis. In particular to synthesis of a mass spectrum cleavable target tyrosine and lysine selective cross-linking agent and identification research of a polypeptide protein structure based on mass spectrum.

Background

Proteins exert their own functions through their three-dimensional structure and protein-protein interactions, and thus, understanding the conformation of a protein is critical to understanding its role in cells and organisms. The main techniques for obtaining information on proteins or protein complexes include X-ray single crystal diffraction, nuclear Magnetic Resonance (NMR) and frozen electron microscopy (cryo-EM). These methods can provide high resolution 3D structural information of proteins. Recently, integrated approaches combining different low resolution techniques have become increasingly important in structural biology, as they can provide supplemental data at different levels. These complementary experimental methods includeResonance Energy Transfer (FRET) (Methods enzymol.2012,504, 371-91), small angle X-ray scattering (SAXS) and Mass Spectrometry (MS) based Methods. The latter methods include natural Mass spectrometry (Nature 2017,541,421-424), hydrogen-deuterium exchange Mass spectrometry (HDX-MS) (Nat. Methods 2019,16,595-602), cross-linked Mass spectrometry (XL-MS) (Anal. Chem.2005,77, 311-318), chemical footprint (assay) methods and ion mobility Mass spectrometry (IM-MS) (Mass Spectrom. Rev.2021,40, 177-200), which combine to provide high resolution protein structural information.

With the continued development of technology, chemical cross-linked mass spectrometry (Chemical cross-linking coupled with mass spectrometry, XL-MS) has been proposed as a powerful tool for studying protein-protein interactions and obtaining information on the three-dimensional structure of proteins and protein complexes at the proteomic level. The technique utilizes chemical cross-linking agents to covalently link two amino acids that are sufficiently closely spaced apart, and then identifies the two amino acid sites that are cross-linked by mass spectrometry (ANALYTICAL CHEMISTRY 2018,90 (1), 144-165). Compared with the traditional protein structure analysis and interaction research technology, the chemical crosslinking mass spectrometry technology has the advantages of high analysis speed, high flux, low requirement on the quantity and purity of protein samples, capability of capturing weak interaction and the like. However, a number of challenges remain in the research to optimize XL-MS technology. The first challenge is that the difficulty of cross-linked mass spectrometry is greatly increased due to the increased search range and poor spectral quality of the cross-linked peptide fragments. To address this limitation, MS cleavable crosslinkers have been developed, such as PIR(Anal.Chem.,2005,77,311–318),DSSO(Mol.Cell.Proteomics,2011,10,M110.002212),DSBU,BuUrBu(Anal.Chem.,2018,90,10990–10999) and DAU (j.am. Soc. Mass spectrum., 2019,30,139-148). The application of the cross-linking agent can simplify the analysis work of the cross-linked mass spectrometry technology and convert the computational complexity from O (n ²) to O (n), thereby producing fewer false positive results. The second challenge is that the interactions between most proteins are weak or transient (Nat Commun,2019,10 (1): 3404), with a large excess of non-crosslinked peptide compared to crosslinked peptide. Most of the cross-linked peptides were not observed by MS due to the randomness of the mass spectrometry experiments. To remove these non-crosslinked peptides, an affinity tag is added to the crosslinking reagent to selectively enrich the crosslinked peptide pairs. For example, crosslinking reagents containing biotin as an affinity handle have been developed (Anal Chem 2022, 94:2713-2722) to enhance detection of crosslinked peptides. Another problem is that most crosslinking agents are directed to only one specific type of residue. For example, N-hydroxysuccinimide (NHS) ester is one of the most widely used crosslinking reagents, reacting mainly with lysine. This particular selective cross-linking of one amino acid residue results in fewer pairs of identifiable cross-linked peptides. In particular, when lysine is not present, the crosslinking analysis effect is not achieved.

Whether the cross-linking arm can break in cross-linked mass spectrometry can also have a significant impact on subsequent mass spectrometry data acquisition and cross-linked peptide fragment identification. If the cross-linking arm contains cleavable bonds that are less energy than the peptide bonds, mass spectrometry breaks the backbone of the peptide fragment while also breaking the cross-linking arm. The mass spectrum cleavable crosslinking agent gradually becomes a research hot spot in the field due to the fact that the mass spectrum cleavable crosslinking agent can reduce the complexity of crosslinked peptide segment identification. The cleavable bonds contained in the crosslinking arms of common mass-cleavable crosslinkers can be broadly divided into two classes, one class being C-S bonds, for example a series of crosslinkers similar to DSSO (Mol Cell Proteomics,2011,10 (1): M110 002212) and CBDPS (Mol Cell Proteomics,2020,19 (4): 624-639); another class is C-N bonds, such as PIR (Anal Chem,2005,77 (1): 311-318) and DSBU crosslinking agents (Anal Chem,2010,82 (16): 6958-6968). In addition to mass-cleavable cross-linking agents, the cross-linking arms of some cross-linking agents can be cleaved by ultraviolet irradiation (Anal Chem,2010,82 (9): 3556-3566), chemical reaction (Protein Sci,2000,9 (8): 1503-1518), and the like, when the cross-linking arms are cleaved, the two peptide fragments of the cross-link are separated from each other, and the problem of identifying cross-linked bipeptides is converted into conventional single peptide identification through analysis of characteristic peaks, so that mass spectrum analysis of cross-linked peptides in complex mixtures is greatly promoted, and the problem of 'n ²' is eliminated.

Lysine-reactive cross-linking agents remain the most widely used reagents in the design and application of cross-linking agents because of the availability of amine-based reaction chemistry and the high abundance of lysine at the interface between protein and PPIs. However, lysine targeting agents by themselves cannot reveal the complete profile of the proteome, as many PPI contact regions lack lysine residues. Thus, the combined XL-MS method using multiple crosslinking chemicals has been applied to enlarge PPI coverage. Recent XL-MS analysis further demonstrated the multiple chemical complementarity to increase PPI coverage by coupling a lysine cross-linker with a carboxyl reactive cross-linker (Mol Cell Proteomics 2021, 20:100084) lysine-cysteine cross-linker (Mol Cell Proteomics 2023, 22:100495) and a cysteine cross-linker (Anal Chem 2023, 95:2532-2539). In general, the continued development of various chemical cross-linking agents and powerful cross-linked search engines remains at a premium for further pushing XL-MS technology to generate a complete map of interactions in cells.

Based on the above studies, the inventors' group of subjects have previously developed several cleavable crosslinking agents, the cleavage being triggerable by a reducing agent, for example, a disulfide-bond-containing crosslinking arm, a DBB crosslinking agent (patent CN 111554345A) and a DBMT crosslinking agent (patent CN 115197156 a) whose reactive groups are urea, a reducing agent being added after the end of the crosslinking, and the type of crosslinking and the crosslinking site being specifically analyzed in comparison of mass spectrum data obtained before and after the reduction. Alternatively, by collision activation in a mass spectrometer, the C-S bond of the sulfoxide can be preferentially cleaved prior to cleavage of the peptide backbone under the influence of Collision Induced Dissociation (CID), thereby physically separating the cross-linked peptide fragments for single sequencing. For example, SBT crosslinkers (patent CN 116574067 a). Notably, such predictable fragments occur independently of crosslinking chemistry, peptide charge, and peptide sequence. These unique properties allow for direct and explicit identification of cross-linked peptides by MS ⁿ analysis in combination with conventional database search tools, thereby further analyzing the cross-linked product. To study protein interactions and elucidate protein structure, the development of various MS cleavable, multi-targeted cross-linking agents is crucial.

Disclosure of Invention

The invention aims to design and develop a heterobifunctional mass spectrum cleavable cross-linking agent (SCT for short) based on the feasibility of clicking an electrochemical reaction on one side to target a tyrosine site and activating ester on one side to target a lysine site and the mass spectrum cleavable property of a C-S bond in a sulfoxide group, so as to realize XL-MS analysis of lysine and tyrosine. The cross-linking agent can realize simultaneous cross-linking of tyrosine and lysine for the first time, and the continuous development of MS cleavable bifunctional cross-linking agents can further promote the development of the application of cross-linking mass spectrometry technology so as to help systematically analyze and clarify the three-dimensional structure of proteins.

The specific technical scheme of the invention is as follows:

A mass spectrum cleavable heterobifunctional crosslinking agent, denoted SCT, is characterized by comprising two different reactive groups, namely a urea group and an N-hydroxysuccinimide group, and a symmetrical mass spectrum cleavable C-S bond as a skeleton structure, and has the following structural general formula:

wherein the R group represents hydrogen, methyl or ethyl.

A preparation method of a mass spectrum cleavable abnormal-shaped bifunctional crosslinking agent comprises the following steps:

Adding cysteine hydrochloride, tert-butyl acrylate and triethylamine into tetrahydrofuran solution according to a molar ratio of 1:1:0.1 at 40 ℃, and stirring overnight; the obtained product and m-chloroperoxybenzoic acid are dissolved in methylene dichloride according to the mol ratio of 1:1, and react for 6 hours at the temperature of 0 ℃; the obtained product is added into ethanol with 1-ethyl-2-phenylhydrazine-1, 2-dicarboxylic acid Ester (EPHD) and triethylamine in the mol ratio of 1:1:2, and the mixture is reacted overnight at 80 ℃; the obtained product and trifluoroacetic acid are dissolved in dichloromethane according to the mol ratio of 1:2.5, and react for 3 hours at room temperature; the obtained product, N-disuccinylcarbonate and triethylamine are added into methylene dichloride according to the mol ratio of 1:1.5:2.2, and stirred overnight to obtain the mass spectrum cleavable heterobifunctional cross-linking agent- - -2, 5-dioxopyrrolidin-1-yl 3- ((2- (3, 5-diketone-1, 2, 4-triazolin-4-yl) ethyl) sulfinyl) propionate, which is abbreviated as SCT.

The application of the mass spectrum cleavable heterobifunctional crosslinking agent is characterized in that a urea group is selectively and specifically crosslinked with tyrosine under the condition of electroclick chemistry, is specifically crosslinked with lysine under the physiological condition, and is used for carrying out chemical crosslinking reaction with protein, carrying out mass spectrum identification on a crosslinked proteolytic product, preliminarily judging a modified peptide fragment in the protein, and when MS ² is carried out fragmentation, the C-S bond in the crosslinking agent is broken at the same time, so that the identification of crosslinked dipeptide is converted into the identification of crosslinked monopeptide, and the crosslinking site in the protein is determined according to the MS ² fragmentation mass spectrum of the crosslinked peptide fragment, thereby identifying the crosslinked product more accurately and rapidly; for the intra-chain crosslinking product, the crosslinking site of the product can be more conveniently identified according to the identified b and y ions containing S or T type crosslinking agent fragments; the method comprises the following specific steps:

(1) Chemical crosslinking reaction: first, specific crosslinking with lysine, SCT was combined with protein according to 20:1 in 100mM PB buffer at pH 7.40, crosslinking at room temperature for 2h; secondly, the protein crosslinked with lysine is crosslinked with tyrosine under the electrochemical condition, namely, the reaction is carried out for 4 hours at room temperature under the voltage of 0.46V, a three-electrode system is used for the electrochemical reaction, namely, a graphite working electrode, a platinum counter electrode and a saturated calomel reference electrode are used for the electrochemical reaction, and the identified protein is angiotensin II, short peptide, ubiquitin protein or glutathione S-transferase protein;

(2) And (3) enzymolysis reaction: when the cross-linked proteins are ubiquitin protein and beta-casein, the product after the cross-linked reaction according to the step (1) is subjected to enzymolysis treatment, trypsin dissolved in 1% acetic acid solution is used, and the mixture is incubated for 4 hours at 37 ℃, wherein the mass ratio of the trypsin to the protein is 1:50;

(3) Mass spectrometry test: analyzing the cross-linked product obtained in the step (1) or the enzymolysis product obtained in the step (2) by using a liquid chromatograph-mass spectrometer, and carrying out liquid phase separation by using a reversed phase column before mass spectrometry, wherein an MS ² spectrogram is generated by collision induced dissociation, and the energy is 15eV; the mass spectrum is operated by using a data dependency acquisition mode DDA, the scanning range of the MS is m/z 200-2000, the temperature of a liquid chromatographic column is kept at 60 ℃, the resolution of the MS is 120000, the AGC target is set as a standard, and the maximum IT is 50MS; the MS ² resolution is 120000, the AGC target is set as standard, the maximum IT is 118MS, the isolation window is 1.2m/s, and the dynamic exclusion is set to 7s; in operation of electrospray ionization source, intrathecal gas flow rate: 40 L.min ^-1, auxiliary gas flow rate: 10 L.min ^-1, spray voltage: 3.8kV, capillary temperature: 325 ℃, auxiliary gas heater temperature: 350 ℃;

(4) Mass spectrometry data analysis: judging a crosslinking site by the mass difference of the peptide fragments after crosslinking and enzymolysis compared with the unmodified peptide fragments;

(5) Determining three-dimensional structural information of protein: and analyzing and sorting the obtained mass spectrum data, and respectively calculating the interval length of the cross-linking agent and the C alpha-C alpha Euclidean distance of tyrosine and lysine in the protein by using Gaussian View 6 software and PyMOL 2.3 software to obtain the three-dimensional structure information of the protein.

The invention has the following advantages:

1. The SCT synthesis step is simple, the raw materials are easy to obtain, the price is low, the environment is protected, and the crosslinking reaction can be completed under the condition of approaching physiology (pH 7.4);

2. The SCT mass spectrum cleavable cross-linking agent designed and synthesized by the invention has the advantages that the cleavage energy of C-S bond in the SCT cross-linking agent is lower than that of amide bond of peptide main chain, and the SCT mass spectrum cleavable cross-linking agent can be broken in a Collision Induced Dissociation (CID) -MS/MS experiment of a mass spectrometer to release characteristic fragment ions. The recognition of the cross-linked dipeptide is converted into a recognition of the cross-linked monopeptide. Since only linear peptides are identified after cleavage by the cross-linker, the secondary search space (n ²) is reduced to a linear search space (2 n).

3. The cross-linking strategy based on the SCT cross-linking agent provided by the invention marks the birth of a new generation of multi-targeting amino acid mass spectrum cleavable cross-linking agent. The method realizes the research on the distance and three-dimensional configuration of tyrosine and lysine in polypeptides and proteins by using a single cross-linking agent, determines the cross-linking site, and promotes the application type research based on the tyrosine and the lysine. The method is helpful for further understanding the three-dimensional structure, interaction and structural dynamics of the protein, and perfects and supplements the research of the polypeptide, the protein and the protein complex by the cross-linked mass spectrometry.

Drawings

FIG. 1 is based on a specific targeted tyrosine and lysine bifunctional crosslinker (SCT) crosslinking strategy.

FIG. 2 mass spectrum of SCT crosslinked angiotensin II.

FIG. 3 mass spectrum of SCT cross-linked polypeptide (FYTPKA).

FIG. 4 mass spectrum of intra-chain crosslinked product of ubiquitin protein.

FIG. 5 spatial structure and crosslinking site of ubiquitin protein.

FIG. 6 mass spectrum of interchain cross-linked product of glutathione S-transferase protein (GST).

FIG. 7 spatial structure and crosslinking site of glutathione S-transferase protein (GST).

Detailed Description

Example 1

The example discloses a preparation method of 2, 5-dioxopyrrolidin-1-yl 3- ((2- (3, 5-dione-1, 2, 4-triazolin-4-yl) ethyl) sulfinyl) propionate, SCT (compound 7) cross-linking agent for short, comprising six steps, the reaction route is as follows:

step one: synthesis of tert-butyl 3- ((2-aminoethyl) thio) propionate (Compound 2):

To a 50ml round bottom flask was added cysteamine hydrochloride (1 eq,2 mmol), THF (1.25 m,1.6 ml) and stirrer; tert-butyl acrylate (1 eq,2 mmol) was then added dropwise to the round-bottomed flask, with stirring, and triethylamine (0.1 eq,0.2 mmol) was added for catalysis; stirring overnight at 40 ℃, the resulting compound was extracted twice with saturated NaHCO ₃ solution, the organic phase was collected and dried over anhydrous Na ₂SO₄, concentrated in vacuo, and purified by silica gel chromatography to give compound 2. The yield is 75％,¹H NMR(400MHz,Chloroform-d)δ2.91(t,J＝6.3Hz,2H),2.75(t,J＝7.3Hz,2H),2.66(t,J＝6.4Hz,2H),2.52(t,J＝7.4Hz,2H),1.46(s,9H).¹³C NMR(101MHz,DMSO-d₆)δ171.04,80.51,45.77,39.00,35.70,28.20,26.53.

Step two: synthesis of tert-butyl 3- ((2-aminoethyl) sulfinyl) propionate (Compound 3):

M-chloroperoxybenzoic acid (1 eq,0.5 mmol) in chloroform was added dropwise to a mixture of compound 2 (1 eq,0.5 mmol) in 15mL of chloroform at 0 ℃, the ice bath was removed and the reaction was stirred at room temperature for 6h. The resulting compound was extracted twice with saturated NaHCO ₃ solution, the organic phase was collected and dried over anhydrous Na ₂SO₄, concentrated in vacuo, and purified by silica gel chromatography to give compound 3. The yield is 83％¹H NMR(400MHz,DMSO-d₆)δ7.33(s,2H),2.94(t,J＝8.7,6.3Hz,2H),2.79(dd,J＝8.6,6.3Hz,2H),2.71(t,J＝7.0Hz,2H),2.51(t,J＝7.0Hz,2H),1.41(s,9H).

Step three: synthesis of ethyl 2- ((2- (3- (tert-butoxy)) -3-propionyl) sulfinyl) ethyl) carbazide-1-carboxylate (Compound 4):

a mixture of triethylamine (0.2 eq,0.2 mmol) and 1-ethyl-2-phenylhydrazine-1, 2-dicarboxylic acid ester (EPHD for short) (1 eq,1 mmol) (1-ethyl-2-phenylhydrazine-1, 2-dicarboxylic acid ester) dissolved in ethanol was added dropwise to compound 3 (2.2 eq,2.2 mmol) dissolved in ethanol at 0℃and stirred for 20 minutes, the ice bath was removed, and the mixture was warmed to 80℃and stirred for 1.5h. The reaction was subjected to two liquid-liquid extractions with 5% aqueous NaHCO ₃. The organic phase was collected, dried over anhydrous Na ₂SO₄, concentrated in vacuo and purified by silica gel chromatography to give compound 4 as a white solid. The yield is 66％,¹H NMR(300MHz,Chloroform-d)δ4.23–4.14(m,2H),3.76(t,2H),3.03(t,J＝14.2,7.8Hz,2H),2.89(t,2H),2.73(t,J＝7.2Hz,2H),1.46(s 9H),1.27(t,J＝7.1,3H).

Step four: synthesis of tert-butyl 3- ((2- (3, 5-dioxo-1, 2, 4-triazolidin-4-yl)) ethyl) sulfinyl) propionate (Compound 5):

In a round bottom flask, potassium hydroxide (2 eq,0.5 mmol) was dissolved in absolute ethanol. Subsequently, compound 4 (1 eq,0.25 mmol) was added to the above solution. The reaction was refluxed at 78℃for 12 hours. The reaction mixture was cooled to room temperature and acidified with hydrochloric acid solution to pH 2.0. The solvent was removed by rotary evaporator and then redissolved in methanol. After filtering off the precipitate, the solution was concentrated in vacuo to give compound 5. The yield is 48％,¹H NMR(400MHz,DMSO-d₆)δ10.01(s,2H),3.35(t,J＝6.3Hz,4H),2.91(t,J＝6.5Hz,4H),1.37(s,9H).

Step five: synthesis of 3- ((2- (3, 5-dioxo-1, 2, 4-triazolidin-4-yl)) ethyl) sulfinyl) propanoic acid (compound 6):

To dried dichloromethane (5 mL) was added 3mmol of t-butyl ester 5 (3 mmol) in stirred solution, 1 part of trifluoroacetic acid (5 mL) was added, stirred at room temperature for 3h, then evaporated and co-evaporated with dichloromethane (4X 50 mL) to give the free acid. The aqueous phase was acidified with hydrochloric acid to ph=1 and extracted 5 times with ethyl acetate. The combined organic layers were dried over MgSO4 and concentrated in vacuo. The crude product (compound 6) was carried on to the next step without further purification.

Step six: synthesis of 2, 5-Dioxopyrrolidin-1-yl 3- ((2- (3, 5-dione-1, 2, 4-triazolin-4-yl) ethyl) sulfinyl) propionate, abbreviated as SCT (Compound 7):

Compound 6, obtained from tert-butyl ester 5 (3 mmol), was dissolved in dichloromethane (50 mL), triethylamine (0.92 mL,6.6 mmol) and N, N-disuccinylcarbonate (1.27 g,4.95 mmol) were added, stirred overnight, evaporated, dissolved in ethyl acetate (100 mL), washed with 5% NaHCO3 (100 mL) and water (100 mL), dried over Na ₂SO₄, concentrated in vacuo to give 2, 5-dioxopyrrolidin-1-yl 3- ((2- (3, 5-dione-1, 2, 4-triazolin-4-yl) ethyl) sulfinyl) propionate (compound 7, the target product SCT). Yield 55％,¹H NMR(300MHz,d-DMSO)δ11.21(s,2H),3.22–3.15(m,4H),2.92(dt,J＝13.5,6.6Hz,2H),2.76(dt,J＝12.9,6.3Hz,2H),2.61(m,4H).¹³C NMR(101MHz,DMSO-d₆)δ170.14,168.49,167.51,45.77,39.00,33.80,26.73.HRMS-ESI(m/z), theoretical number: c ₁₁H₁₄N₄O₇S₁([M+H]⁺) 347.0617, experimental measurement 347.0621

The cross-linking strategy using the bifunctional cross-linker (SCT) of the present invention is shown in figure 1.

Example 2

Crosslinking identification of model polypeptide angiotensin II

(1) Chemical crosslinking reaction: first, an electrochemical crosslinking tyrosine reaction was performed, and angiotensin II (1 eq,0.2 mM) and SCT (10 eq,2 mM) were dissolved in 100mM PB buffer pH 7.4, and reacted at room temperature for 4 hours under a voltage of 0.46V. Secondly, carrying out chemical crosslinking lysine reaction, firstly, specifically crosslinking with lysine, dissolving an SCT crosslinking agent and protein in 50mM PB buffer solution with pH of 7.5, and crosslinking for 1h at room temperature;

(2) The crosslinked product was analyzed by an Agilent 1290 affinity liquid chromatograph-Bruker micrOTOF-QII mass spectrometer (LC-MS ⁿ). Liquid phase separation was performed using an Agilent Zorbax300SB-C18 reverse phase column (4.6X105 mm,5 μm, column temperature 40 ℃) prior to mass spectrometry. The flow rate is 1mL/min; the linear gradient is: the mobile phase buffer solution A and the mobile phase buffer solution B are respectively water and acetonitrile containing 0.1% of formic acid, wherein the mobile phase buffer solution A is 5% of B in 0-5min, the mobile phase buffer solution B is 5-60% of B in 6-55min and 60-98% of B in 56-60 min. The MS ² spectrum was generated by Collision Induced Dissociation (CID), energy 15eV, and mass spectrum data shown in figure 2.

(3) Mass spectrometry data analysis: if the mass of the peptide fragment after cleavage by the crosslinking enzyme is 233.07Da higher than the mass of the unmodified peptide fragment, then the lysine crosslinked to the peptide fragment is considered to be a chain-end crosslinked peptide; likewise, if the mass of a cross-linked peptide fragment is 345.31Da than the mass of an unmodified peptide fragment, then the tyrosine cross-linked to that peptide fragment is considered to be a chain-end cross-linked peptide. The inter-and intra-chain crosslinks are the result of two reactive groups in SCT reacting with tyrosine and lysine in the peptide fragment, respectively, and the peptide fragment mass will increase by 232.07Da. As the name suggests, if the two-step crosslinking reaction occurs in tyrosine and lysine in one peptide segment, it is considered as intra-chain crosslinking; if one SCT molecule linked tyrosine and lysine is located in two peptide fragments, it is considered to be inter-chain crosslinking; when the secondary mass spectrum fragmentation data is analyzed, the b and y ions with the mass increased by 159.01Da indicate that the ion contains S-shaped fragments of the cross-linking agent SCT, and the b and y ions with the mass increased by 54.21Da indicate that the ion contains T-shaped fragments of the cross-linking agent SCT.

(4) The results of the above data indicate that specific crosslinking of tyrosine in angiotensin II was achieved by cross-linking reaction of angiotensin II using SCT as bifunctional cross-linker, and identifying 1 chain end cross-linking site information altogether (mass spectral data as shown in fig. 2).

Example 3

Cross-linking identification of model polypeptides (FYTPKA)

(1) Chemical crosslinking reaction: a chemical crosslinking lysine reaction was performed, and polypeptide (FYTPKA) (1 eq,0.2 mM) and SCT (10 eq,2 mM) were dissolved in 100mM PB buffer pH 7.4, and the crosslinking reaction was performed for 2h at room temperature without applying a voltage.

(2) The crosslinked product was analyzed by an Agilent 1290 affinity liquid chromatograph-Bruker micrOTOF-QII mass spectrometer (LC-MS ⁿ). Liquid phase separation was performed using an Agilent Zorbax 300SB-C18 reverse phase column (4.6X105 mm,5 μm, column temperature 40 ℃) prior to mass spectrometry. The flow rate is 1mL/min; the linear gradient is: the mobile phase buffer solution A and the mobile phase buffer solution B are respectively water and acetonitrile containing 0.1% of formic acid, wherein the mobile phase buffer solution A is 5% of B in 0-5min, the mobile phase buffer solution B is 5-60% of B in 6-55min and 60-98% of B in 56-60 min. The MS ² spectrum was generated by Collision Induced Dissociation (CID) with an energy of 15eV. Mass spectrometry data are shown in figure 2.

(3) Mass spectrometry data analysis: the same as angiotensin II.

(4) The results of the above data indicate that specific crosslinking of lysine in polypeptide (FYTPKA) was achieved by chemically crosslinking polypeptide (FYTPKA) using SCT as mass-cleavable lysine selective crosslinker, and identifying 1 chain end crosslinking site information altogether (mass spectral data is shown in fig. 3).

Example 4

Three-dimensional structural identification of ubiquitin proteins

(1) Chemical crosslinking reaction: first, electrochemical crosslinking tyrosine reaction was performed, ubiquitin protein (1 eq,0.2 mM) and SCT (10 eq,2 mM) were dissolved in 100mM PB buffer pH 7.4, and reacted at room temperature for 4h under a voltage of 0.46V. Secondly, carrying out chemical crosslinking lysine reaction, dissolving an SCT crosslinking agent and protein in 50mM PB buffer solution with pH of 7.5, and crosslinking for 2 hours at room temperature;

(2) And (3) enzymolysis reaction: the solution was subjected to an enzymatic hydrolysis reaction using trypsin dissolved in 1% acetic acid solution, and incubated at 37℃for 4 hours at a trypsin to protein mass ratio of 1:50.

(3) Mass spectrometry test: the enzymatic hydrolysis products were analyzed by an Agilent 1290 Infinicity liquid chromatograph-Bruker micrOTOF-QII mass spectrometer (LC-MS ⁿ). Liquid phase separation was performed using an Agilent Zorbax 300SB-C18 reverse phase column (1.0X105 mm,5 μm, column temperature 40 ℃) prior to mass spectrometry. The flow rate is 1mL/min; the linear gradient is: the mobile phase buffer solution A and the mobile phase buffer solution B are respectively water and acetonitrile containing 0.1% of formic acid, wherein the mobile phase buffer solution A is 5% of B in 0-5min, the mobile phase buffer solution B is 5-60% of B in 6-55min and 60-98% of B in 56-60 min. The MS ² spectrum was generated by Collision Induced Dissociation (CID) with an energy of 15eV.

(4) Mass spectrometry data analysis: and performing electroclick chemical crosslinking reaction of the tyrosine residues in the SCT targeted ubiquitin, and then performing enzymolysis analysis on the crosslinked product. The results showed that not only was the mass spectrum peak of the trypsin cleaved product (TLSDYNIQK, m/z 541.28 ²⁺) detected, but also the m/z 713.35 ²⁺ mass spectrum peak, which was shifted by 345.31Da compared to the uncrosslinked intact peptide (TLSDYNIQK, m/z 541.28 ²⁺), initially thought that SCT crosslinked tyrosine in peptide fragment (TLSDYNIQK). From the CID characteristic ion fragment of m/z 715.35 ²⁺, the crosslinking site was identified as Y ⁵⁹. The m/z 656.31 ²⁺ mass spectrum peak was also detected, with a mass shift of 233.07Da compared to the uncrosslinked intact peptide (TLSDYNIQK, m/z 541.28 ²⁺), and the Y ion binding of b, Y ion containing S, T and A type crosslinker fragments generated by cleavage of C-S bonds by HCD high energy collision dissociation, resulted in fragmentation of the crosslinked peptide fragment in the MS ² mass spectrum, demonstrated that the intra-chain crosslinked product crosslinking site was Y ⁵⁹-K⁶³. In addition, a crosslinking reaction of lysine in SCT-crosslinked ubiquitin was performed and the crosslinked product was subjected to enzymatic analysis. The cleavage product of trypsin crosslinked by SCT was detected (LIFAGK, m/z648.35 ⁺) with a mass shift of 233.31Da compared to the intact uncrosslinked peptide fragment m/z 881.57 ⁺. From the characteristic ion fragment of m/z 881.57 ⁺, the crosslinking site was identified as K ⁴⁸. (mass spectrometry data are shown in FIG. 4).

(5) Determining three-dimensional structural information of protein: the gap length of the SCT crosslinker and the distance contributed by the tyrosine and lysine side chains were calculated using Gaussian View 6 software. The C.alpha. -C.alpha.Euclidean distance of ubiquitin protein was calculated by PyMOL 2.3 software on PDB files (website: http:// www.rcsb.org /). The three-dimensional structure and the crosslinking site of ubiquitin protein are shown in FIG. 4 and FIG. 5.

Example 5

Spatial structure characterization of glutathione S-transferase protein (GST)

(1) Chemical crosslinking reaction: first, an electrochemical crosslinking tyrosine reaction was performed, GST protein (1 eq,0.2 mM) and SCT (10 eq,2 mM) were dissolved in 100mM PB buffer pH7.4, and reacted at 0.46V for 4h at room temperature. Secondly, carrying out chemical crosslinking lysine reaction, dissolving an SCT crosslinking agent and protein in 50mM PB buffer solution with pH of 7.5, and crosslinking for 2 hours at room temperature;

(2) The solution was subjected to an enzymatic hydrolysis reaction using trypsin dissolved in 1% acetic acid solution, and incubated at 37℃for 4 hours at a trypsin to protein mass ratio of 1:50.

(3) The above crosslinked enzymatic products were analyzed using a Vanquish UPLC with Orbitrap Fusion Tribrid in combination with a mass spectrometer (LC-MS ⁿ). Liquid phase separation was performed using an ACQUITY PREMIER CSH C-18 reverse phase chromatography column (5 μm,1.0x 150mm,Waters) prior to mass spectrometry. The liquid chromatography column temperature was maintained at 60 ℃. Mass spectra were run using DDA, MS scan range from m/z 200-2000. The resolution of MS ¹ is 120000, the agc target is set to standard, and the maximum IT is 50MS. The MS ² resolution was 60000, the AGC target was set to standard, the maximum IT was 118MS, and the isolation window was 1.2m/z. The dynamic exclusion is set to 7s. During operation of electrospray ionization source ESI, intrathecal gas flow rate: 40 L.min ^-1, auxiliary gas flow rate: 10 L.min ^-1, spray voltage: 3.8kV, capillary temperature: 325 ℃, auxiliary gas heater temperature: 350 ℃.

(4) Mass spectrometry data analysis: identical to ubiquitin protein.

(5) The gap length of the SCT crosslinker and the distance contributed by the tyrosine and lysine side chains were calculated using Gaussian View 6 software. The Cα -Cα Euclidean distance of GST protein was calculated by PyMOL 2.3 software for PDB files (website: http:// www.rcsb.org /). The spatial structure and crosslinking site of GST protein are shown in FIG. 6 and FIG. 7.

Claims (3)

1. A mass spectrum cleavable heterobifunctional crosslinking agent, denoted SCT, is characterized by comprising two different reactive groups, namely a urea group and an N-hydroxysuccinimide group, and a symmetrical mass spectrum cleavable C-S bond as a skeleton structure, and has the following structural general formula:

wherein the R group represents hydrogen, methyl or ethyl.

2. A method of preparing a mass spectrum cleavable heterobifunctional crosslinking reagent of claim 1, comprising the steps of:

Adding cysteine hydrochloride, tert-butyl acrylate and triethylamine into tetrahydrofuran solution according to a molar ratio of 1:1:0.1 at 40 ℃, and stirring overnight; the obtained product and m-chloroperoxybenzoic acid are dissolved in methylene dichloride according to the mol ratio of 1:1, and react for 6 hours at the temperature of 0 ℃; the obtained product is added into ethanol with 1-ethyl-2-phenylhydrazine-1, 2-dicarboxylic acid ester and triethylamine in the mol ratio of 1:1:2, and the reaction is carried out overnight at 80 ℃; the obtained product and trifluoroacetic acid are dissolved in dichloromethane according to the mol ratio of 1:2.5, and react for 3 hours at room temperature; the obtained product, N-disuccinylcarbonate and triethylamine are added into methylene dichloride according to the mol ratio of 1:1.5:2.2, and stirred overnight to obtain the mass spectrum cleavable heterobifunctional cross-linking agent- - -2, 5-dioxopyrrolidin-1-yl 3- ((2- (3, 5-diketone-1, 2, 4-triazolin-4-yl) ethyl) sulfinyl) propionate, which is abbreviated as SCT.

3. The application of the mass spectrum cleavable heterobifunctional crosslinking agent of claim 1, wherein the urea azole group is selectively and specifically crosslinked with tyrosine under the electro-click chemistry condition, is specifically crosslinked with lysine under the physiological condition, uses the bifunctional crosslinking agent to carry out chemical crosslinking reaction with protein, carries out mass spectrum identification on a crosslinked proteolytic product, preliminarily judges a modified peptide in the protein, breaks a C-S bond in the crosslinking agent while breaking a crosslinked peptide when MS ² breaks, converts the recognition of crosslinked dipeptide into the recognition of crosslinked monopeptide, and determines a crosslinking site in the protein according to a MS ² broken mass spectrum of the crosslinked peptide, thereby more accurately and rapidly recognizing the crosslinked product; for the intra-chain crosslinking product, the crosslinking site of the product can be more conveniently identified according to the identified b and y ions containing S or T type crosslinking agent fragments; the method comprises the following specific steps:

(1) Chemical crosslinking reaction: first, specific crosslinking with lysine, SCT was combined with protein according to 20:1 in 100mM PB buffer at pH 7.40, crosslinking at room temperature for 2h; secondly, the protein crosslinked with lysine is crosslinked with tyrosine under the electrochemical condition, namely, the reaction is carried out for 4 hours at room temperature under the voltage of 0.46V, a three-electrode system is used for the electrochemical reaction, namely, a graphite working electrode, a platinum counter electrode and a saturated calomel reference electrode are used for the electrochemical reaction, and the identified protein is angiotensin II, short peptide, ubiquitin protein or glutathione S-transferase protein;

(2) And (3) enzymolysis reaction: when the cross-linked proteins are ubiquitin protein and beta-casein, the product after the cross-linked reaction according to the step (1) is subjected to enzymolysis treatment, trypsin dissolved in 1% acetic acid solution is used, and the mixture is incubated for 4 hours at 37 ℃, wherein the mass ratio of the trypsin to the protein is 1:50;

(3) Mass spectrometry test: analyzing the cross-linked product obtained in the step (1) or the enzymolysis product obtained in the step (2) by using a liquid chromatograph-mass spectrometer, and carrying out liquid phase separation by using a reversed phase column before mass spectrometry, wherein an MS ² spectrogram is generated by collision induced dissociation, and the energy is 15eV; the mass spectrum is operated by using a data dependency acquisition mode DDA, the scanning range of the MS is m/z 200-2000, the temperature of a liquid chromatographic column is kept at 60 ℃, the resolution of the MS is 120000, the AGC target is set as a standard, and the maximum IT is 50MS; the MS ² resolution is 120000, the AGC target is set as standard, the maximum IT is 118MS, the isolation window is 1.2m/s, and the dynamic exclusion is set to 7s; in operation of electrospray ionization source, intrathecal gas flow rate: 40 L.min ^-1, auxiliary gas flow rate: 10 L.min ^-1, spray voltage: 3.8kV, capillary temperature: 325 ℃, auxiliary gas heater temperature: 350 ℃;

(4) Mass spectrometry data analysis: judging a crosslinking site by the mass difference of the peptide fragments after crosslinking and enzymolysis compared with the unmodified peptide fragments;

(5) Determining three-dimensional structural information of protein: and analyzing and sorting the obtained mass spectrum data, and respectively calculating the interval length of the cross-linking agent and the C alpha-C alpha Euclidean distance of tyrosine and lysine in the protein by using Gaussian View 6 software and PyMOL 2.3 software to obtain the three-dimensional structure information of the protein.