A method based on solid-phase glycoprotein t antigen glycopeptide enrichment and enzymatic analysis

Abstract

The application discloses a method based on solid-phase glycoprotein T antigen glycopeptide enrichment and enzyme cutting analysis, which comprises the following steps: extracting protein from a sample; obtaining polypeptide through proteolysis; oxidizing galactose and N-acetyl galactose; fixing with hydrazide resin and cutting with PNGase F enzyme; treating with galactoside enzyme and performing mass spectrometry analysis. The method obtains three structures, T antigen glycopeptide of core 1, core 2 glycopeptide and core 8 glycopeptide. The T antigen glycopeptide and the core 2 glycopeptide are distinguished through mass spectrometry analysis, and the T antigen glycopeptide and the core 8 are distinguished through different glycosidases. The method can enrich and distinguish glycopeptides containing T antigens, core 2 and core 8, and has important significance for finding specific markers with T antigen modification in tumor tissues and body fluids, research on early diagnosis and prognosis markers of diseases and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomolecular analysis reagent technology, specifically relating to a method for the enrichment and enzyme digestion analysis of solid-phase glycoprotein T antigen glycopeptides (SPTAgE). Background Technology

[0002] The T antigen is a disaccharide structure formed by galactose binding to acetylgalactosamine via a β(1-3) linker, belonging to the core 1 structure of common O-glycans. T antigen expression can be detected in 90% of human cancer types. While T antigens are hidden in normal cells, they are selectively exposed on the surface of cancer cells in breast, colon, prostate, and bladder cancers. The T antigen is a truncated O-glycan, small in size and simple in structure. It has a non-physiological glycan structure in the human body, thus it can be recognized as a foreign substance by the immune system. T antigen tests in most cancer patients can detect cancer before any biopsy reveals it. Because the T antigen is a protein on the surface of blood and skin cells, it can be recognized by immune system antibodies, thus serving as a disease biomarker for diagnosis or prognosis. However, few methods are available for detailed identification of intact proteins modified by T antigens in cancer, especially in the early stages; simultaneously, the core 8 structure, similar in structure to the T antigen, can interfere with the acquisition of information on T antigen modification sites.

[0003] Therefore, it is necessary to develop a method based on solid-phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis to specifically identify T antigen modification sites and their related glycoproteins. Summary of the Invention

[0004] The purpose of this invention is to provide a method based on solid-phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis (SPTAgE), which can distinguish between core 2 and core 8 O-glycosylated glycoproteins or glycopeptides, thereby solving the problem of identifying T antigen glycosylation sites and enriching related glycoproteins in the prior art.

[0005] One technical solution of the present invention is:

[0006] A method based on solid-phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis includes the following steps:

[0007] (1) Extract proteins from the sample;

[0008] (2) Protein hydrolysis to obtain polypeptides;

[0009] (3) Oxidation of galactose and N-acetylgalactose;

[0010] (4) Immobilization with acylhydrazide resin and PNGase F digestion;

[0011] (5) Galactosidase treatment and mass spectrometry analysis.

[0012] Furthermore, in step (1), the sample includes: a tissue sample or a body fluid sample.

[0013] Furthermore, in step (2), the polypeptide obtained by proteolytic hydrolysis includes:

[0014] ① Add dithiothreitol to the protein solution and react at 37℃ for 1-1.5 hours;

[0015] ② Add iodoacetamide and react in a dark room at room temperature for 0.5-1 hour to obtain the sample;

[0016] ③ Dilute the sample by adding ammonium bicarbonate until the final concentration of the ammonium bicarbonate is 90-110 mM, and the pH value of the sample is between 7 and 9;

[0017] ④ Add sequencing-grade trypsin, gently shake, and hydrolyze at 37°C for 16-18 hours to obtain a polypeptide solution;

[0018] ⑤ Add formic acid to the polypeptide solution until the pH value is adjusted to 2-3;

[0019] ⑥ Pre-treat the C18 extraction column by adding the polypeptide solution to the C18 extraction column, adding the filtrate back to the C18 extraction column, washing the C18 extraction column multiple times with 0.1% TFA, and finally eluting the polypeptide with 50-60% acetonitrile containing 0.1% TFA, and repeating the process multiple times.

[0020] ⑦ Combine the eluted peptides and freeze-dry them under vacuum to obtain purified peptides.

[0021] Furthermore, in step (3), the oxidation of galactose and N-acetylgalactose includes: adding dimethyl sulfoxide, sodium phosphate buffer, horseradish peroxidase and galactose oxidase to the purified polypeptide, and then reacting at a temperature of 35°C for 0.9-1.1 hours to obtain glycopeptides.

[0022] Furthermore, the volume ratio of dimethyl sulfoxide, sodium phosphate buffer, horseradish peroxidase, and galactose oxidase is 9-11:22-23:12-13:4-6.

[0023] Furthermore, in step (4), the fixation of the acylhydrazine resin and the PNGase F enzyme digestion include: the glycopeptide is covalently bound to the acylhydrazine resin, then PNGase F enzyme is added, and after reacting at a temperature of 37°C for 3 hours, the supernatant is removed and the resin is obtained by washing.

[0024] Furthermore, in step (5), the galactosidase treatment includes:

[0025] ① Add deionized water, 50-60mM sodium phosphate buffer and β-galactosidase to the resin and react at 37°C for 1 hour to sever the link between galactose and acetylgalactosamine in the T antigen, thereby obtaining a polypeptide containing the T antigen and the core 2 modification site.

[0026] Furthermore, in step (5), the galactosidase treatment further includes:

[0027] ② Add α-galactosidase to the resin treated with β-galactosidase to cleave the link between galactose and acetylgalactosamine in galactose-α(1-3)-acetylgalactosamine, and obtain a polypeptide containing core 8.

[0028] Furthermore, the volume ratio of the deionized water, sodium phosphate buffer, and β-galactosidase is 156-160:38-42:1.8-2.2.

[0029] Furthermore, in step (5), the mass spectrometry analysis includes: analyzing the polypeptide containing T antigen and core 2 modification site and the polypeptide containing core 8 respectively using liquid chromatography-mass spectrometry to obtain primary and secondary mass spectra, and using bioinformatics software to analyze the mass spectrometry data to obtain site information.

[0030] This invention provides a method for enriching and analyzing T antigen-modified glycopeptides based on solid-phase glycoprotein T antigen glycopeptides. This method can specifically enrich and analyze T antigen-modified glycopeptides from complex protein peptides, and has wide applications in various scenarios (including but not limited to): qualitative and quantitative analysis of T antigen modifications in normal and cancer cells; qualitative and quantitative analysis of T antigen modifications in clinical body fluids and tissues; and qualitative and quantitative analysis of glycopeptides modified with intact T antigens. Therefore, this method can enrich and distinguish glycopeptides containing T antigen, core 2, and core 8, which is of great significance for discovering specific biomarkers with T antigen modifications in tumor tissues and body fluids, as well as for research on early cancer diagnosis and prognostic biomarkers. Attached Figure Description

[0031] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1 This is a schematic diagram of the workflow for enriching glycopeptides modified with T antigen in this invention.

[0033] Figure 2 This is a schematic diagram illustrating the enzymatic hydrolysis of proteins and the oxidation of glycopeptides by galactose oxidase (GAO) in this invention.

[0034] Figure 3 This is a schematic diagram illustrating the oxidation mechanism of galactose and N-acetylgalactose by galactose oxidase (GAO) in this invention.

[0035] Figure 4 This is a schematic diagram illustrating the covalent bonding process between oxidized galactose and N-acetylgalactose glycopeptides and hydrazide resin in this invention.

[0036] Figure 5 This is a schematic diagram of the solid-phase enzyme enrichment preparation of T antigen glycopeptides in this invention. Detailed Implementation

[0037] This invention develops a method based on solid-phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis (SPTAgE), comprising the following steps:

[0038] Step 1: Sample protein extraction

[0039] Method 1: Protein extraction from tissue samples

[0040] Freeze the tissue sample in dry ice or at -80°C for 2-3 hours, then grind the tissue.

[0041] Add 400-600 μL of RIPA lysis buffer to the homogenized tissue, and use an ultrasonic homogenizer (30-40% energy) to homogenize for 30 seconds. After that, place the sample on ice to cool. Repeat this step 4-6 times. After centrifugation at 12,000×g for 10 minutes, collect the supernatant.

[0042] Take 2-4 microliters of sample, dilute it 5-10 times, and use BCA to test the protein concentration;

[0043] Dissolve 800-1000 micrograms of protein in 400-600 microliters of urea, depending on the concentration. The final urea concentration is 8M. Gently shake the sample to ensure complete protein dissolution and obtain a protein solution.

[0044] Method 2: Protein extraction from body fluid samples (blood as an example)

[0045] The collected blood samples were allowed to coagulate at room temperature (usually 15-30 minutes). Then, the samples were centrifuged at 1,000-2,000 × g at 4°C for 10 minutes, and the supernatant (serum) was collected. Serum protein concentration was determined using a micro spectrophotometer.

[0046] Take 20 μL of serum sample and add 180 μL of urea directly (final concentration 8M);

[0047] The protein solution was obtained by heat denaturation at 90°C for 10 minutes.

[0048] Step 2: Protein hydrolysis

[0049] Please see Figure 2 , Figure 2 This is a schematic diagram illustrating the protein enzymatic hydrolysis and glycopeptide oxidation via galactose oxidase (GAO) in this invention. Figure 2 As shown, protein hydrolysis (trypsin) yields polypeptides and glycopeptides. GAO can oxidize N-acetylgalactosamine (GalNAc) and galactose (Gal) to form aldehyde groups. Therefore, the specific steps of protein hydrolysis are as follows:

[0050] Add 1 / 10 volume of 120mM dithiothreitol (DTT) to the protein solution extracted from tissues or body fluids, and react at 37°C for 1-1.5 hours;

[0051] Add 1 / 10 volume of 160mM iodoacetamide and react in a dark room at room temperature for 0.5-1 hour;

[0052] Dilute the sample 5-6 times, add freshly prepared 1M ammonium bicarbonate, and the final ammonium bicarbonate concentration is 90-110mM. The pH of the test sample is between 7 and 9.

[0053] Add 20-25 micrograms of sequencing-grade trypsin, gently shake, and react at 37°C for 16-18 hours to hydrolyze and obtain the peptide.

[0054] Add 10-12% formic acid (w / v) to the solution until the pH is adjusted to 2-3;

[0055] After pretreating the C18 extraction column, add the sample, add the filtrate back to the extraction column, wash the extraction column 5-6 times (1.0-1.2 mL) with 0.1% TFA, and elute the peptides with 400-500 μL of 50-60% acetonitrile (ACN) containing 0.1% TFA. Repeat the last step 2-3 times (all ratios are volume ratios).

[0056] The washed peptides were combined and then freeze-dried under vacuum to obtain purified peptides.

[0057] Step 3: Oxidation of galactose and N-acetylgalactose

[0058] Please see Figure 3 , Figure 3 This is a schematic diagram illustrating the oxidation mechanism of galactose and N-acetylgalactose by galactose oxidase (GAO) in this invention. Figure 3 As shown, 18-22 μL of dimethyl sulfoxide, 44-46 μL of sodium phosphate buffer (pH=7), 24-26 μL of horseradish peroxidase and 8-12 μL of galactose oxidase were added to the purified peptides, and then the mixture was reacted at 35°C for 0.9-1.1 hours.

[0059] Step 4: Immobilization with acylhydrazide resin and digestion with PNGase F

[0060] Please see Figure 4 , Figure 4 This is a schematic diagram illustrating the covalent bonding process between oxidized galactose and N-acetylgalactose glycopeptides and the hydrazide resin in this invention. Figure 4 As shown, the glycopeptides oxidized by galactose oxidase were covalently bound to acylhydrazine resin, and then 1 μL of PNGase F enzyme was added. After reacting at 37°C for 3 hours, the supernatant was removed and the resin was washed.

[0061] Step 5: Galactosidase treatment and mass spectrometry analysis

[0062] Please see Figure 5 , Figure 5 This is a schematic diagram illustrating the solid-phase enzyme enrichment preparation of T antigen glycopeptides in this invention. Figure 5 As shown, β-galactosidase was used to enzymatically digest the T antigen glycopeptide and core 2 glycopeptide on a solid phase; α-galactosidase was used to obtain the core 8 glycopeptide. Mass spectrometry detection can distinguish the T antigen glycopeptides of core 2 and core 1. Details are as follows:

[0063] Add 156-160 μL of deionized water, 38-42 μL of 50-60 mM sodium phosphate buffer (pH 7-8), and 1.8-2.2 μL of β-galactosidase to the resin treated with PNGase F enzyme, and react at 37°C for 1 hour to remove the link between galactose and acetylgalactosamine in the T antigen, to obtain a polypeptide containing T antigen (core 1) and core 2 modification sites.

[0064] The peptides containing T antigen modification sites obtained from enzymatic digestion were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain primary and secondary mass spectra. The chromatographic method was 10-50% ACN, and the mass spectrometric energy was CE 30. The mass spectrometry data were analyzed using bioinformatics software. The T antigen modification sites showed an increase of 203 Da (GalNAc-peptide), while the non-modification sites remained unchanged. The core 2-glycopeptide has a GlcNAc-GalNAc-peptide structure, while the T antigen glycopeptide is a GalNAc-peptide. Therefore, the mass spectrometry detection showed that the core 2-glycopeptide had a 406 Da content.

[0065] The resin treated with β-galactosidase was then further treated with α-galactosidase, which cleaved the link between galactose and acetylgalactosamine in the galactose-α(1-3)-acetylgalactosamine (core 8 structure), yielding a polypeptide containing core 8. Mass spectrometry analysis and software interpretation under the same conditions were then performed to obtain the site information of galactose-α(1-3)-acetylgalactosamine modification, thus distinguishing it from T antigen modification.

[0066] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments. However, the present invention is not limited to the listed embodiments, but should also include any other known modifications within the scope of the claims of the present invention.

[0067] First, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places throughout this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0068] Secondly, this invention is described in detail using structural diagrams, etc. When detailing the embodiments of this invention, for ease of explanation, the diagrams may be partially enlarged, deviating from the general scale. Furthermore, the diagrams are merely examples and should not limit the scope of protection of this invention. In addition, actual manufacturing should include three-dimensional space with length, width, and depth.

[0069] Example 1

[0070] Enrichment of T antigen-modified proteins in the urine or tissues of pancreatic cancer patients and healthy subjects.

[0071] Cancer can affect the O-glycosylation of certain proteins, which may eventually be secreted into the urine of patients. Therefore, this invention can identify specific T antigen-modified proteins in the urine of pancreatic cancer patients.

[0072] (1) Urine sample collection and processing

[0073] Each participant collected 10 mL of urine, which was completely dried in a freeze dryer. The dried urine sample was reconstituted with 1 mL of deionized water, and ethanol was added at a volume ratio of 1:5 to precipitate proteins. The sample was then centrifuged at 12,000 × g and 4 °C for 10 minutes, and the supernatant was collected. The urine protein concentration was determined using the Bradford method.

[0074] (2) Urinary protein hydrolysis and peptide purification

[0075] Add 300 μL of 8M urea solution to 100 μL of urine protein solution (10 μg / μL) and heat denature at 90°C for 10 minutes;

[0076] Add 40 μL of 120 mM dithiothreitol (DTT) and react at 37°C for 1–1.5 hours;

[0077] Add 44 μL of 160 mM iodoacetamide and react in the dark at room temperature for 0.5-1 hour;

[0078] Dilute the sample volume to 1.6 ml, add 40 μL of freshly prepared 1M ammonium bicarbonate, and adjust the pH of the test sample to be between 7 and 9.

[0079] Add 20 micrograms of sequencing-grade trypsin, gently shake, and react at 37°C for 16-18 hours to hydrolyze and obtain the peptide.

[0080] Add 65 μL of 10% formic acid to the solution to adjust the pH to 2-3;

[0081] After pretreating the C18 extraction column (1 mL volume), add the sample, add the filtrate back to the extraction column, wash the extraction column 5-6 times with 0.1% TFA (1 mL each time), and elute the peptide 3 times with 500 μL of 50% acetonitrile (ACN) containing 0.1% TFA.

[0082] The washed peptides were combined and then freeze-dried under vacuum to obtain purified peptides.

[0083] (3) Oxidation of galactose and N-acetylgalactose

[0084] Add 20 μL of dimethyl sulfoxide, 45 μL of sodium phosphate buffer (pH=7), 25 μL of horseradish peroxidase, and 10 μL of galactose oxidase to the dried peptide sample, and then react at 35°C for 1 hour.

[0085] (4) Immobilization with acylhydrazide resin and PNGase F digestion

[0086] In the previous step, the glycopeptides oxidized by galactose oxidase covalently bind to the acylhydrazine resin. Then, 1 μL of PNGase F enzyme is added, and the reaction is carried out at 37°C for 3 hours. After that, the supernatant is removed and the resin is washed.

[0087] (5) Galactosidase treatment and mass spectrometry analysis

[0088] After the resin was treated with PNGase F enzyme in the previous step, 158 μL of deionized water, 40 μL of 50-60 mM sodium phosphate buffer and 2 μL of β-galactosidase were added, and the mixture was reacted at 37°C for 1 hour to sever the link between galactose and acetylgalactosamine in the T antigen.

[0089] The peptides containing T antigen modification sites obtained from enzymatic digestion were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain primary and secondary mass spectra. The chromatographic method was 10-50% ACN, and the mass spectrometric energy was CE 30. The mass spectrometry data were analyzed using bioinformatics software. The T antigen modification sites showed an increase of 203 Da (GalNAc-peptide), while the non-modification sites remained unchanged. The core 2-glycopeptide has a GlcNAc-GalNAc-peptide structure, while the T antigen glycopeptide is a GalNAc-peptide. Therefore, the mass spectrometry detection showed that the core 2-glycopeptide had a 406 Da content.

[0090] The resin treated with β-galactosidase was then further treated with α-galactosidase, which cleaved the link between galactose and acetylgalactosamine in the galactose-α(1-3)-acetylgalactosamine (core 8 structure), yielding a polypeptide containing core 8. Mass spectrometry analysis and software interpretation under the same conditions were then performed to obtain the site information of galactose-α(1-3)-acetylgalactosamine modification, thus distinguishing it from T antigen modification.

[0091] This invention enriches T antigen-modified glycopeptides in pancreatic cancer and normal samples, then labels the glycopeptides with the isotope tag IBT-10, and finally analyzes the LC-MS / MS data using MaxQuant software. This allows for the quantitative analysis of the expression differences of T antigen-modified proteins between pancreatic cancer patients at different stages and healthy subjects, thereby developing specific glycoprotein biomarkers.

[0092] Example 2

[0093] The expression of T antigen-modified proteins in pancreatic cancer cells and normal pancreatic cells is abnormally elevated. Therefore, this invention can identify specific T antigen-modified proteins in pancreatic cancer cells and study the affected signaling pathways by inhibiting the biosynthesis of T antigen.

[0094] (1) Culture of pancreatic cells

[0095] The normal pancreatic cell line HTERT-HPNE and two pancreatic cancer cell lines MIA PaCa-2 and AsPC-1 were divided into groups at a ratio of 1×10⁴ cells / cm². 2 The cells were seeded at a density of [insert density here] in 6-well plates. After the cells adhered, the medium was replaced with serum-free medium and 2.5 μM itraconazole was added to treat the cells for 48 hours.

[0096] (2) Extraction of cellular proteins

[0097] After cell processing, remove the culture medium from the 6-well plate and wash the cells twice with PBS. Add 500 μL of RIPA lysis buffer (containing 50 μL of 100× protease inhibitor and 50 μL of 100× phosphatase inhibitor) to each well. Collect the cells into EP tubes using a cell scraper, and sonicate on ice for 30 seconds, then cool on ice for 30 seconds. Repeat this process 6 times. Centrifuge the EP tubes at 12,000 rpm at 4°C for 15 minutes and collect the supernatant. Determine protein concentration using the BCA method.

[0098] (3) Cell protein digestion and peptide purification

[0099] Add 300 μL of 8M urea solution to 100 μL of cell protein solution (1 μg / μL) and heat denature at 90°C for 10 minutes;

[0100] Add 40 μL of 120 mM dithiothreitol (DTT) and react at 37°C for 1–1.5 hours;

[0101] Add 44 μL of 160 mM iodoacetamide and react in the dark at room temperature for 0.5-1 hour;

[0102] Dilute the sample volume to 1.6 ml, add 40 μL of freshly prepared 1M ammonium bicarbonate, and adjust the pH of the test sample to be between 7 and 9.

[0103] Add 20 micrograms of sequencing-grade trypsin, gently shake, and react at 37°C for 16-18 hours to hydrolyze and obtain the peptide.

[0104] Add 65 μL of 10% formic acid to the solution to adjust the pH to 2-3;

[0105] After pretreating the C18 extraction column (1 mL volume), add the sample, add the filtrate back to the extraction column, wash the extraction column 5-6 times with 0.1% TFA (1 mL each time), and elute the peptide 3 times with 500 μL of 50% acetonitrile (ACN) containing 0.1% TFA.

[0106] The washed peptides were combined and then freeze-dried under vacuum to obtain purified peptides.

[0107] (4) Oxidation of galactose and N-acetylgalactose

[0108] Add 20 μL of dimethyl sulfoxide, 45 μL of sodium phosphate buffer (pH=7), 25 μL of horseradish peroxidase, and 10 μL of galactose oxidase to the dried peptide sample, and then react at 35°C for 1 hour.

[0109] (5) Immobilization with acylhydrazide resin and PNGase F digestion

[0110] In the previous step, the glycopeptides oxidized by galactose oxidase covalently bind to the acylhydrazine resin. Then, 1 μL of PNGase F enzyme is added, and the reaction is carried out at 37°C for 3 hours. After that, the supernatant is removed and the resin is washed.

[0111] (6) Galactosidase treatment and mass spectrometry analysis

[0112] After the resin was treated with PNGase F enzyme in the previous step, 158 μL of deionized water, 40 μL of 50-60 mM sodium phosphate buffer and 2 μL of β-galactosidase were added, and the mixture was reacted at 37°C for 1 hour to sever the link between galactose and acetylgalactosamine in the T antigen.

[0113] The peptides containing T antigen modification sites obtained from enzymatic digestion were analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) to obtain primary and secondary mass spectra. The chromatographic method was 10-50% ACN, and the mass spectrometric energy was CE 30. The mass spectrometry data were analyzed using bioinformatics software. The T antigen modification sites showed an increase of 203 Da (GalNAc-peptide), while the non-modification sites remained unchanged. The core 2-glycopeptide has a GlcNAc-GalNAc-peptide structure, while the T antigen glycopeptide is a GalNAc-peptide. Therefore, the mass spectrometry detection showed that the core 2-glycopeptide had a 406 Da content.

[0114] The resin treated with β-galactosidase was then further treated with α-galactosidase, which cleaved the link between galactose and acetylgalactosamine in the galactose-α(1-3)-acetylgalactosamine (core 8 structure), yielding a polypeptide containing core 8. Mass spectrometry analysis and software interpretation under the same conditions were then performed to obtain the site information of galactose-α(1-3)-acetylgalactosamine modification, thus distinguishing it from T antigen modification.

[0115] By inhibiting the biosynthesis of T antigen in pancreatic cancer cells and then analyzing the changes in T antigen-modified proteins and other affected proteins and signaling pathways, we can not only improve our understanding of the mechanisms related to the occurrence and development of pancreatic cancer, but also make significant contributions to the discovery of drug targets for pancreatic cancer treatment.

[0116] In summary, compared with existing technologies, this invention provides a method based on solid-phase glycoprotein T antigen glycopeptide enrichment and enzymatic digestion analysis (SPTAgE). First, biological or clinical samples are subjected to protein extraction via lysis. Then, the protein is hydrolyzed using a protease to obtain peptides. Next, the samples are treated with galactose oxidase (GAO), causing the glycans on the glycopeptides to oxidize and bind to an acylhydrazine resin. Finally, PNGase F is used to cleave the N-glycans from the peptides. (See also...) Figure 1 , Figure 1 This is a schematic diagram of the workflow for enriching glycopeptides modified with T antigen in this invention. Figure 1 As shown, this method yields three structures: a core 1 T antigen glycopeptide obtained by treating a galactose-β(1-3)-acetylgalactosamine (T antigen) glycopeptide with β-galactosidase; a core 2 glycopeptide obtained by enzymatic hydrolysis of a galactose-β(1-3)-acetylgalactosamine glycopeptide containing acetylglucosamine with β-galactosidase; and a core 8 glycopeptide obtained by enzymatic hydrolysis of a galactose-α(1-3)-acetylgalactosamine (core 8 structure) with α-galactosidase. The T antigen glycopeptide and core 2 glycopeptide are distinguished by mass spectrometry, while the T antigen glycopeptide and core 8 glycopeptide are distinguished by different glycosidases. This method can enrich and differentiate glycopeptides containing T antigen, core 2, and core 8, which is of great significance for discovering specific biomarkers modified with T antigen in tumor tissues and body fluids, as well as for research on early cancer diagnosis and prognostic biomarkers.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for enrichment and enzyme digestion analysis of solid-phase glycoprotein T antigen glycopeptides, characterized in that, Including the following steps: (1) Extract proteins from the sample; (2) Obtaining polypeptides through proteolytic digestion: ① Add dithiothreitol to the protein solution and react at 37°C for 1-1.5 hours; ② Add iodoacetamide and react in a dark room at room temperature for 0.5-1 hour to obtain the sample; ③ Dilute the sample by adding ammonium bicarbonate until the final concentration of the ammonium bicarbonate is 90-110 mM, and the pH value of the sample is between 7 and 9; ④ Add sequencing-grade trypsin, gently shake, and hydrolyze at 37°C for 16-18 hours to obtain a polypeptide solution; ⑤ Add formic acid to the polypeptide solution until the pH value is adjusted to 2-3; ⑥ Pre-treat the C18 extraction column by adding the polypeptide solution to the C18 extraction column, adding the filtrate back to the C18 extraction column, washing the C18 extraction column multiple times with 0.1% TFA, and finally eluting the polypeptide with 50-60% acetonitrile containing 0.1% TFA, and repeating the process multiple times. ⑦ Combine the eluted peptides and freeze-dry them under vacuum to obtain purified peptides; (3) Oxidation of galactose and N-acetylgalactose: Dimethyl sulfoxide, sodium phosphate buffer, horseradish peroxidase and galactose oxidase were added to the purified polypeptide, and the mixture was reacted at 35°C for 0.9-1.1 hours to obtain glycopeptides, wherein the volume ratio of dimethyl sulfoxide, sodium phosphate buffer, horseradish peroxidase and galactose oxidase was 9-11:22-23:12-13:4-6; (4) Immobilization with acylhydrazide resin and PNGase F digestion; (5) Galactosidase treatment and mass spectrometry analysis: ① Add deionized water, 50-60 mM sodium phosphate buffer and β-galactosidase to the resin, and react at 37°C for 1 hour to cleave the link between galactose and acetylgalactosamine in the T antigen, to obtain a polypeptide containing the T antigen and the core 2 modification site. The volume ratio of the deionized water, sodium phosphate buffer and β-galactosidase is 156-160:38-42:1.8-2.

2. ② Add α-galactosidase to the resin treated with β-galactosidase to cleave the link between galactose and acetylgalactosamine in galactose-α(1-3)-acetylgalactosamine, and obtain a polypeptide containing core 8.

2. The method for enrichment and enzyme digestion analysis of solid-phase glycoprotein T antigen glycopeptides according to claim 1, characterized in that, In step (1), the sample includes: a tissue sample or a body fluid sample.

3. The method for enrichment and enzymatic digestion analysis of solid-phase glycoprotein T antigen glycopeptides according to claim 1, characterized in that: In step (4), the fixation of the acylhydrazine resin and the PNGase F enzyme digestion include: the glycopeptide is covalently bound to the acylhydrazine resin, then PNGase F enzyme is added, the reaction is carried out at a temperature of 37°C for 3 hours, the supernatant is removed, and the resin is obtained by washing.

4. The method for enrichment and enzyme digestion analysis of solid-phase glycoprotein T antigen glycopeptides according to claim 1, characterized in that, In step (5), the mass spectrometry analysis includes: analyzing the peptide containing T antigen and core 2 modification site and the peptide containing core 8 respectively by liquid chromatography-mass spectrometry to obtain primary and secondary mass spectra, and parsing the mass spectrometry data with bioinformatics software to obtain site information.

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