Protein cysteine carboxyethylation modification detection method

Abstract

The invention provides a method for detecting cysteine carboxyethylation modification of protein, and relates to the technical field of protein modification omics. According to the method disclosed by the invention, a cysteine carboxyethylation modified peptide fragment is used as an antigen to prepare a high-specificity cysteine carboxyethylation modified universal antibody; carrying out affinity enrichment on a carboxyethylation modified peptide fragment in a biological sample by utilizing the prepared cysteine carboxyethylation modified ubiquitous antibody; and finally, identifying carboxyethylation modification sites, polypeptide sequences and substrate proteins of the enriched peptide fragments by using mass spectrometry. Compared with the existing cystine carboxyethylation modification detection method, the method has the advantages of high specificity, low false positive rate and the like, can directly indicate protein cysteine carboxyethylation modification sites in various biological samples, and has simple and feasible operability. The invention provides an effective means for high-throughput detection of protein cysteine carboxyethylation modification in a biological sample.

Description

Technical Field

[0001] The present invention relates to the technical field of protein modification omics, and in particular to a method for detecting protein cysteine ​​carboxyethylation modification. Background Art

[0002] Cysteine ​​carboxyethylation is one of the important forms of protein post-translational modification. It is a stable post-translational modification of protein cysteine ​​formed by the covalent bonding of the metabolite 3-hydroxypropionic acid (3-HPA) with the cysteine ​​side chain thiol to form a thioether bond. This modification can change the structure and function of proteins, affect cellular protein degradation and signal transduction, and induce the production of neoantigens. It is an important cause of the occurrence and development of autoimmune diseases. Specific and high-throughput screening of cysteine ​​carboxyethylation-modified proteins in biological samples is crucial for analyzing the biological processes regulated by cysteine ​​carboxyethylation.

[0003] The traditional method for detecting cysteine ​​carboxyethylation-modified proteins is based on labeling proteins with radioactive 3-HPA salts, and detecting the degree of isotope labeling by autoradiography to indicate the cysteine ​​carboxyethylation modification of proteins. However, this method has problems such as radioactive pollution, cumbersome operation, high cost, and low sensitivity. In addition, this method can only be used to detect cysteine ​​carboxyethylation-modified proteins in living cells, and cannot be used to analyze cysteine ​​carboxyethylation modification of proteins in tissue samples and body fluids. Based on the above technical background, the present invention intends to provide a method for high specificity, simple operation, no radioactive pollution, and high-throughput detection of cysteine ​​carboxyethylation-modified proteins in various biological samples. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the deficiencies of the prior art, the present invention provides a method for detecting protein cysteine ​​carboxyethylation modification based on the combined use of a specific anti-cysteine ​​carboxyethylation pan-antibody and liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0008] In the first aspect, the present invention provides a pan-antibody that specifically targets the carboxyethylation modification of cysteine ​​in proteins, wherein the pan-antibody is prepared by covalently modifying 3-hydroxypropionic acid to the thiol group of cysteine ​​via a thioether bond to obtain an antigenic peptide containing the carboxyethylation modification, coupling the antigenic peptide to a carrier protein KLH, and then immunizing New Zealand white rabbits to obtain a pan-antibody that is resistant to the carboxyethylation modification.

[0009] Specifically, the antigen peptide contains 11 amino acid residues, and the amino acid sequence of the antigen peptide is CXXXXC(hpa)XXXXX, wherein C represents cysteine, and X represents a mixture of 19 amino acids other than cysteine, and the 7th amino acid residue of the antigen peptide is carboxyethyl-modified cysteine ​​C (hpa).

[0010] In a second aspect, the present invention provides a method for detecting carboxyethylation of cysteine ​​in a protein, the method specifically comprising:

[0011] (1) preparing pan-antibodies modified with cysteine ​​carboxyethylation;

[0012] (2) Enriching cysteine ​​carboxyethylated proteins in biological samples such as cells or tissues by immunoprecipitation or affinity chromatography using cysteine ​​carboxyethylated pan-antibodies;

[0013] (3) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify and analyze the cysteine ​​carboxyethylation modification sites of the enriched proteins.

[0014] (III) Beneficial effects

[0015] The present invention innovatively uses carboxyethylated peptides as antigens and successfully prepares a highly specific pan-antibody for carboxyethylated cysteine. By using this antibody that can accurately identify carboxyethylated proteins, effective detection of the carboxyethylated state of proteins is achieved. Compared with existing flux analysis methods, this method exhibits higher specificity and lower false positive rate, can directly mark carboxyethylated proteins, and is suitable for complex biological samples from different sources. In addition, when combined with mass spectrometry analysis technology, this method can efficiently and accurately identify carboxyethylated cysteine ​​proteins in biological samples, and is easy to operate and highly practical. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Dot immunoblotting was used to detect the specificity of the pan-antibody modified with cysteine ​​carboxyethylation.

[0017] Figure 2 Western blot was used to detect the specificity of carboxyethylated cysteine ​​modified proteins in cervical cancer Hela cell samples using a pan-antibody to detect carboxyethylated cysteine.

[0018] Figure 3Mass spectrometry identification of peptides of cysteine ​​carboxyethylated proteins enriched in 3-hydroxypropionic acid treated samples. (A) MS analysis of peptide SINNDTTYC(hpa)IK of protein LACB2(Q53H82). (B) MS analysis of peptide THEAEIVEGENHTYC(hpa)IR of protein FLNA(P21333). (C) MS analysis of peptide DHVFLC(hpa)EGEEPK of protein JUPI2(Q9H910). (D) MS analysis of peptide AVLFC(hpa)LSEDKK of protein COF1(P23528). (E) MS analysis of peptide GKDYYC(hpa)ILGIEK of protein DNJB4(Q9UDY4). (F) MS analysis of the peptide HLNEIDLFHC(hpa)IDPNDSK of protein TEBP(Q15185). (G) MS analysis of the peptide DVPEDVAQEMVESGYVC(hpa)EGDHK of protein WNK1(Q9H4A3). (H) MS analysis of the peptide FELSC(hpa)YSLAPQIK of protein ASSY(P00966). (I) MS analysis of the peptide DDVFLSVPC(hpa)ILGQNGISDLVK of protein LDHA(P00338). DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Example 1

[0021] Preparation of immune antigen peptide library

[0022] The peptide solid phase synthesis method was used to obtain an antigen peptide containing carboxyethyl modification (CXXXXC(hpa)XXXXX) and an unmodified control peptide (CXXXXCXXXXX), and the peptide was coupled to a carrier protein. C represents cysteine, X represents a mixture of 19 amino acids other than cysteine, and the 7th amino acid residue of the antigen peptide containing carboxyethyl modification is carboxyethyl modified cysteine ​​C (hpa).

[0023] In this embodiment, the coupling scheme of the synthetic antigen peptide and the carrier protein is as follows:

[0024] Synthesize 5 mg of an antigenic peptide (CC) containing only two cysteines without carboxyethyl modification and 9 mg of an antigenic peptide (CC(hpa)) containing only two cysteines with carboxyethyl modification; couple them to mcKLH, represented by C-KLH and C(hpa)-KLH, respectively; specifically, follow the following steps:

[0025] (1) KLH protein was dissolved in PBS buffer to a final concentration of 10 mg / mL;

[0026] (2) MBS was dissolved in DMF to a final concentration of 10 mg / mL. KLH and MBS were mixed at a volume ratio of 10:1, incubated at room temperature for 30 min, and then passed through a PD-10 chromatography column;

[0027] (3) Slowly add the sample, elute with PB buffer, and collect the activated KLH in an EP tube; dissolve CC or CC(hpa) in PBS and mix with activated KLH in equal volumes, incubate at room temperature for 3 h, and dialyze at 4°C overnight.

[0028] Example 2

[0029] Animal Free and Protein Cysteine ​​Carboxyethylation Modification Pan-Antibody Production

[0030] 1. Animal immunization

[0031] (1) Select healthy 6-8 week old New Zealand white rabbits.

[0032] (2) Take 500 μL of 0.8 mg / mL C(hpa)-KLH antigen peptide and mix it with Freund's complete adjuvant to form a stable emulsion. Inject the emulsion into the rabbit's body, subcutaneously at multiple points on the rabbit's back and groin. Every 2 weeks, take 250 μL of 0.8 mg / mL antigen with appropriate adjuvant and method for boosting immunization.

[0033] (3) One week after the fourth immunization, 1 mL of serum was collected and the serum titer was tested by ELISA. If the serum titer was greater than 30,000, subsequent purification could be continued; if the serum titer was less than 30,000, immunization could be continued.

[0034] 2. ELISA to identify antiserum antibody titer and specificity

[0035] C-KLH and C(hpa)-KLH antigen peptides were coated as antigens at a concentration of 2 μg / ml, and purified antiserum was diluted with blocking solution (starting at 1:100), and ELISA was performed to compare the OD values ​​of C-KLH and C(hap)-KLH after reaction with antiserum to determine the effect of antiserum. The results showed that the C(hap)-KLH antigen-embedded group had a significant immune response, while the unmodified peptide (C-KLH)-embedded group had no significant immune response (Table 1), indicating that the anti-cysteine ​​carboxyethyl modified antiserum is effective.

[0036] Table 1 ELISA identification of antiserum antibody titer results

[0037] Rabbit serum dilution ratio C-KLH C(hap)-KLH 1:10 0.08 1.96 1:50 0.03 0.85 1:100 0.02 0.28 Blank 0.01 0.01

[0038] 3. Polyclonal antibody purification

[0039] Positive screening using antigen affinity purification and negative screening using an unmodified peptide library:

[0040] (1) 2 mg of CC(hpa) antigen peptide CXXXXC(hpa)XXXXX was covalently linked to a Sepharose 4B column activated with hydrogen bromide to prepare a carboxyethylated affinity purification column. Similarly, 2 mg of CC antigen peptide (CXXXXCXXXXX) was covalently linked to a Sepharose 4B column activated with hydrogen bromide to prepare an unmodified peptide affinity adsorption column.

[0041] (2) Incubate 10 mL of anti-cysteine ​​carboxyethylated antiserum with the carboxyethylated affinity purification column overnight.

[0042] (3) Pre-wash with HCl at pH 5.0 to remove contaminating antibodies.

[0043] (4) Elution was performed using a 0.15 M glycine buffer at pH 2.5 and rapid neutralization was performed after elution to prepare affinity purified antibodies.

[0044] (5) The affinity-purified antibody is passed through an unmodified peptide affinity adsorption column to remove the antibody against the unmodified peptide and obtain a specific cysteine ​​carboxyethylated pan-antibody.

[0045] (6) Elute the BSA affinity adsorption column using pH = 2.5, 0.15 M glycine buffer and dialyze with PBS buffer for replacement.

[0046] (7) Ultrafiltration antibody concentration was reduced to 1-2 mg / ml, and the pan-antibody modified by cysteine ​​carboxyethylation was denoted as panC-hpa.

[0047] (8) ELISA was used to detect the titer of affinity purified antibodies.

[0048] (9) Dot blot hybridization was used to detect the potency and specificity of affinity purified antibodies.

[0049] The specific binding of the pan-antibody to three different peptide libraries was detected by dot immunoblotting. The amount of each peptide library was as follows: Figure 1 As shown in the figure; each peptide library contains 11 amino acid residues, where C represents cysteine, X represents a mixture of 19 amino acids other than cysteine, and the seventh amino acid residue in the peptide library is unmodified cysteine ​​(C), lactylated lysine (Kla) or carboxyethylated cysteine ​​(Chpa). The results show that the prepared cysteine ​​carboxyethylated pan-antibody can only be immunohybridized with the CXXXXC(hpa)XXXXX antigen peptide, indicating that the prepared cysteine ​​carboxyethylated pan-antibody has high specificity and strong recognition ability for cysteine ​​carboxyethylated peptides.

[0050] The affinity-purified cysteine ​​carboxyethylated pan-antibody was gradiently diluted, and the titer and specificity of the cysteine ​​carboxyethylated pan-antibody were detected by ELISA. The results are shown in Table 2. When the dilution ratio was small, the prepared cysteine ​​carboxyethylated pan-antibody had a strong immune reaction with the C(hap)-KLH antigen. The cysteine ​​carboxyethylated pan-antibody needs to be used in a gradient of 1:200 to 1:1000.

[0051] Table 2 ELISA identification of affinity purified antibody titer results

[0052]

[0053]

[0054] After affinity-purified cysteine ​​carboxyethylated pan-antibody was diluted 1:1000, 0, 10, 100 μL / mL of CC(hpa) antigen peptide were added for binding experiments, and the titer of cysteine ​​carboxyethylated pan-antibody was detected by ELISA kit embedded with C(hap)-KLH antigen peptide. The results are shown in Table 3. The immune reaction of the prepared cysteine ​​carboxyethylated pan-antibody with C(hap)-KLH antigen was significantly inhibited by the competitive binding of the added CC(hpa) peptide, indicating that the prepared cysteine ​​carboxyethylated pan-antibody has a strong recognition ability for free cysteine ​​carboxyethylated peptide.

[0055] Table 3 ELISA test results of pan-modified antibodies after antigen peptide binding

[0056] CC(hap) peptide (μL / mL) C(hap)-KLH 0 0.82 10 0.63 100 0.32

[0057] Example 3

[0058] Detection of cysteine ​​carboxyethylated proteins in cervical cancer Hela cells using pan-antibody modified with cysteine ​​carboxyethylation

[0059] The western blot method was used to detect the carboxyethylated proteins in cervical cancer Hela cells using the pan-antibody for carboxyethylated cysteine ​​prepared in Example 2, which includes the following steps:

[0060] (1) Cell treatment and collection: Hela cells in the logarithmic phase were treated with PBS solution, 1mM, 10mM, 50mM, and 100mM 3-HPA solution, respectively. After 6 hours of drug treatment, the cell culture supernatant was discarded and the Hela cells were collected using a cell scraper.

[0061] (2) Cell lysis and protein quantification: 1×10 7 To each Hela cell, add 100 μL of cell lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 5 mM EDTA, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM Na3VO4, 0.5 ug / ml eupeptin), mix well and lyse the cells in an ice bath for 30 min; after high-speed centrifugation for 10 min (12000 g × 4°C), collect the cell lysis supernatant and quantify the protein by the BCA method.

[0062] (3) Immunoblotting experiment: 100 μg of protein from each group was taken for 10% polyacrylamide gel electrophoresis, and the gel protein band was transferred to a PVDF membrane (Millipore) using a BioRad electrotransfer system. After blocking the unbound sites on the membrane with 5% skim milk powder in TBS-T blocking solution, the membrane was incubated overnight at 4°C with a cysteine ​​carboxyethylated pan-modified antibody (1:500 dilution); after washing the unbound pan-modified antibody with TBS-T solution three times, a goat anti-rabbit secondary antibody (Thermo Scientific) diluted in TBS-T solution (1:5000) was added and incubated at room temperature for 1 hour with shaking; after washing the hybridized membrane with TBS-T solution three times, an ECL ultrasensitive colorimetric solution (Thermo Scientific) was added, and the color image was captured under ultraviolet light using an ImageQuant ECL instrument (GE).

[0063] The results are as follows Figure 2As shown in the figure, the proteins extracted from the whole cells of cervical cancer Hela cells were separated by SDS-PAG gel electrophoresis, transferred to the membrane, and hybridized with the cysteine ​​carboxyethylation modified pan-antibody, showing obvious positive bands; the results showed that when the same amount of protein was loaded, the protein sample treated with 3-hydroxypropionic acid had an obvious signal, and the signal intensity was significantly enhanced with the increase of the 3-hydroxypropionic acid treatment concentration, indicating that the cysteine ​​carboxyethylation modified pan-antibody can effectively detect cysteine ​​carboxyethylation modified proteins.

[0064] Example 4

[0065] Enrichment of cysteine ​​carboxyethylated proteins in cervical cancer Hela cells by using pan-antibody modified with cysteine ​​carboxyethylation

[0066] (1) Cell treatment and collection: After treating Hela cells in the logarithmic phase with 100 mM 3-HPA solution for 6 hours, discard the cell culture supernatant and collect the Hela cells using a cell scraper. 7 To each Hela cell, add 200 μL of immunoprecipitation lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 5 mM EDTA, 2 mM sodium pyrophosphate, 25 mM β-glycerophosphate, 1 mM Na3VO4, 0.5 ug / mL leupeptin), mix well and lyse the cells in an ice bath for 30 min; after high-speed centrifugation for 15 min (12000 g × 4°C), collect the cell lysis supernatant.

[0067] (2) Immunoprecipitation: The collected cell supernatant was diluted to 10 mL with immunoprecipitation lysis buffer, and 30 μg of cysteine ​​carboxyethylated pan-antibody was added. The immunoprecipitate was shaken at 4°C for 3 hours. 0.5 mL of Dynabeads magnetic beads (ThermoScientific) suspension was added, and the immunoprecipitate was shaken at 4°C for 30 minutes. After low-speed centrifugation to precipitate the magnetic bead complex, the Dynabeads magnetic bead mixture was washed 5 times with immunoprecipitation lysis buffer.

[0068] (3) Protein elution: Use 0.15 M glycine at pH 2.5 to elute the cysteine ​​carboxyethylated proteins enriched by Dynabeads magnetic beads for subsequent protein spectrum identification.

[0069] Example 5

[0070] Identification of carboxyethylated cysteine ​​sites in proteins by protein profiling

[0071] (1) Protein extraction, enzyme digestion, purification and desalting: The enriched cysteine ​​carboxyethylated proteins were added to RIPA lysis buffer, mixed and lysed on ice for 30 minutes. Subsequently, the supernatant was collected by low-temperature high-speed centrifugation, 5 volumes of acetone were added, and precipitation was carried out at -80°C for 1 hour. The supernatant was discarded by low-temperature high-speed centrifugation; the precipitate was washed twice with 80% acetone, 50mM NH4HCO3 solution was added, and the protein precipitate was re-dissolved by ultrasound. After the protein concentration was determined by BCA, trypsin was added at a ratio of 50:1, and the enzyme digestion was carried out at 37°C overnight. The enzymatic peptides were desalted using a C18 Cartridge column, vacuum-dried, and stored at -80°C for later use.

[0072] (2) LC-MS / MS analysis: 1 μg of peptide sample was chromatographically separated by EASY-nLC 1200 liquid chromatography system (ThermoScientific), and peptide mass spectrometry data was detected by mass spectrometer (Thermo Scientific). Buffer: Solution A is 0.1% formic acid in water, and solution B is 0.1% formic acid in acetonitrile (80% acetonitrile). The chromatographic column was balanced with 100% solution A. The sample was gradient separated by reverse phase C18 column (75 μm × 15 cm) at a flow rate of 350 nl / min. The separated sample was analyzed by DDA mass spectrometry using a mass spectrometer. Detection mode: positive ion. Primary mass spectrometry scanning range: 350-2000 m / z, resolution: 60000@m / z 200, AGC target: 3e6, Maximum IT: 50 ms. Secondary mass spectrometry acquisition method: After each full scan, the top 20 parent ions in the primary spectrum are fragmented and the corresponding secondary mass spectra are collected. Secondary mass spectrometry resolution: 15000@m / z 200, AGC target: 1e5, Maximum IT: 45ms, Activity Type: HCD, Normalized collis ion energy: 28.

[0073] (3) Database search: The RAW files of the mass spectrometer were imported into pFind3 (V2.8) software for searching carboxyethylated cysteine. The protein sequence library relied on the Uniprot (Release 2023_05) database.

[0074] Figure 3The mass spectrometry identification spectra of cysteine ​​carboxyethylated proteins are shown. The results show that the B / Y ions of cysteine ​​carboxyethylated proteins can be identified in the fragmentation ion spectra of the peptides of proteins LACB2, FLNA, JUPI2, COF1, DNJB4, TEBP, WNK1, ASSY, and LDHA, indicating the reliability of mass spectrometry identification of cysteine ​​carboxyethylated proteins enriched by cysteine ​​carboxyethylated pan-antibodies.

[0075] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pan-antibody specifically targeting protein cysteine ​​carboxyethylation modification, characterized in that: The pan-antibody is prepared by covalently modifying 3-hydroxypropionic acid to the thiol group of cysteine ​​via a thioether bond to obtain an antigenic peptide containing carboxyethyl modification, coupling the antigenic peptide to a carrier protein KLH, and then immunizing New Zealand white rabbits to obtain a pan-antibody resistant to carboxyethyl modification.

2. A pan-antibody specifically targeting protein cysteine ​​ethylation modification according to claim 1, characterized in that: The antigen peptide comprises 11 amino acid residues, and the amino acid sequence of the antigen peptide is CXXXXC(hpa)XXXXX, wherein C represents cysteine, and X represents a mixture of 19 amino acids other than cysteine, and the 7th amino acid residue of the antigen peptide is carboxyethyl-modified cysteine ​​C (hpa).

3. A method for detecting carboxyethylation of protein cysteine, characterized in that: The method is specifically as follows: (1) preparing pan-antibodies modified with cysteine ​​carboxyethylation; (2) Enriching cysteine ​​carboxyethylated proteins in biological samples such as cells or tissues by immunoprecipitation or affinity chromatography using cysteine ​​carboxyethylated pan-antibodies; (3) Liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to identify and analyze the cysteine ​​carboxyethylation modification sites of the enriched proteins.

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