CN114720543A - Method for enriching and enzyme digestion analysis of T antigen glycopeptide based on solid phase glycoprotein - Google Patents
Method for enriching and enzyme digestion analysis of T antigen glycopeptide based on solid phase glycoprotein Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
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Abstract
The invention discloses a method for enriching and enzyme digestion analysis of a glycopeptide based on a solid phase glycoprotein T antigen, which comprises the following steps: extracting a protein from the sample; carrying out proteolysis to obtain polypeptide; oxidation of galactose and N-acetylgalactosamine; fixing hydrazide resin and performing enzyme digestion with PNGase F; galactosidase treatment and mass spectrometry. The method obtains three structures, namely T antigen glycopeptide of core 1, core 2 glycopeptide and core 8 glycopeptide. The T antigen glycopeptide and core 2 glycopeptides are distinguished by mass spectrometry, whereas the T antigen glycopeptide is distinguished from core 8 by different glycosidases. The method can enrich and distinguish glycopeptides containing T antigen, core 2 and core 8, and has important significance in finding specific markers with T antigen modification in tumor tissues and body fluids, and research on early diagnosis and prognosis markers of diseases.
Description
Technical Field
The invention belongs to the technical field of biomolecule analysis reagents, and particularly relates to a method for enriching and enzyme digestion analysis (SPTAgE) of solid phase glycoprotein T antigen glycopeptide.
Background
The T antigen is a disaccharide structure formed by combining galactose and acetylgalactosamine through beta (1-3) connection, and belongs to a core 1 structure in common O-glycan. T antigen expression is detectable in 90% of human cancer types. The T antigen is hidden in normal cells, but selectively exposed on the surface of cancer cells in breast, colon, prostate and bladder cancers. The T antigen is a truncated O-glycan, and has small volume and simple structure. It has a non-physiological glycan structure in the human body, so it can be recognized as a foreign body by the immune system. The T antigen test of most cancer patients can be detected before any biopsy reveals cancer. Since T antigen is a protein on the surface of blood and skin cells and can be recognized by immune system antibodies, it can be used as a disease biomarker for diagnosis or prognosis. However, few methods are available for detailed identification of complete protein modification by T antigen in cancer, especially at early stages; meanwhile, the core 8 structure similar to the T antigen structure can interfere the acquisition of the T antigen modification site information.
Therefore, there is a need to develop a method based on enrichment and enzyme digestion analysis of solid phase glycoprotein T antigen glycopeptide to specifically identify T antigen modification sites and their associated glycoproteins.
Disclosure of Invention
The invention aims to provide a method for enriching and enzyme digestion analysis (SPTAgE) based on a solid phase glycoprotein T antigen glycopeptide, which can distinguish core 2 and core 8O-glycosylated glycoprotein or glycopeptide at the same time so as to solve the problems of identifying T antigen glycosylation sites and enriching related glycoprotein in the prior art.
The invention has a technical scheme that:
a method based on solid phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis comprises the following steps:
(1) extracting a protein from the sample;
(2) carrying out proteolysis to obtain polypeptide;
(3) oxidation of galactose and N-acetylgalactosamine;
(4) fixing hydrazide resin and performing enzyme digestion with PNGase F;
(5) galactosidase treatment and mass spectrometry.
Further, in step (1), the sample comprises: tissue samples or body fluid samples.
Further, in step (2), the obtaining of the polypeptide by proteolysis includes:
adding dithiothreitol into the protein solution, and reacting for 1-1.5 hours at the temperature of 37 ℃;
adding iodoacetamide, and reacting in a dark room at room temperature for 0.5-1 hour to obtain a sample;
thirdly, diluting the sample, adding ammonium bicarbonate until the final concentration of the ammonium bicarbonate is 90-110mM and the pH value of the sample is between 7 and 9;
adding sequencing-grade trypsin, slightly oscillating, and hydrolyzing at 37 ℃ for 16-18 hours to obtain a polypeptide solution;
adding formic acid into the polypeptide solution until the pH value is reduced to 2-3;
sixthly, pretreating the C18 extraction column, adding the polypeptide solution into the C18 extraction column, adding the filtrate into the C18 extraction column again, washing the C18 extraction column with 0.1% TFA for a plurality of times, finally, eluting the polypeptide with 50-60% acetonitrile containing 0.1% TFA, and repeating the steps for a plurality of times;
and seventhly, combining the eluted polypeptides, and carrying out vacuum freeze drying to obtain the purified polypeptide.
Further, in the step (3), the galactose and N-acetylgalactosamine oxidation includes: adding dimethyl sulfoxide, sodium phosphate buffer solution, horseradish peroxidase and galactose oxidase into the purified polypeptide, and reacting at 35 ℃ for 0.9-1.1 h to obtain the glycopeptide.
Further, the volume ratio of the dimethyl sulfoxide to the sodium phosphate buffer solution to the horseradish peroxidase to the galactose oxidase is 9-11: 22-23: 12-13: 4-6.
Further, in the step (4), the cleavage of the hydrazide resin immobilization with PNGase F comprises: covalently bonding the glycopeptide and hydrazide resin, adding PNGase F enzyme, reacting at 37 ℃ for 3 hours, removing supernatant, and cleaning to obtain the resin.
Further, in the step (5), the galactosidase treatment comprises:
adding deionized water, 50-60mM sodium phosphate buffer solution and beta galactosidase into the resin, and reacting for 1 hour at the temperature of 37 ℃ to cut off the connection between galactose and acetylgalactosamine in the T antigen, thereby obtaining the polypeptide containing the T antigen and the core 2 modified site.
Further, in the step (5), the galactosidase treatment further comprises:
② adding alpha galactosidase into the resin after beta-galactosidase treatment to cut off the connection between galactose and acetylgalactosamine in galactose-alpha (1-3) -acetylgalactosamine, and obtaining the polypeptide containing core 8.
Further, the volume ratio of the deionized water to the sodium phosphate buffer to the beta-galactosidase is 156-160: 38-42: 1.8-2.2.
Further, in the step (5), the mass spectrometry comprises: respectively analyzing the polypeptide containing the T antigen and the core 2 modification site and the polypeptide containing the core 8 by using liquid chromatography-mass spectrometry to obtain primary and secondary mass spectra, and analyzing mass spectrum data by using bioinformatics software to obtain site information.
The invention provides a method for enriching and enzyme digestion analysis of T antigen glycopeptide based on solid phase glycoprotein, which can specifically enrich and analyze T antigen modified glycopeptide from complex protein polypeptide and has wide application in the following various scenes (including but not limited to the scenes): qualitative and quantitative analysis of T antigen modification in normal and cancer cells; qualitative and quantitative analysis of T antigen modification in clinical body fluids and tissues; the qualitative and quantitative analysis of complete T antigen modified glycopeptide makes it possible to enrich and distinguish glycopeptide containing T antigen, core 2 and core 8, and has important significance in finding T antigen modified specific marker in tumor tissue and body fluid, early diagnosis of cancer, research of prognosis marker, etc.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein the content of the first and second substances,
FIG. 1 is a schematic diagram of the process of the present invention for enriching glycopeptides containing T antigen modifications;
FIG. 2 is a schematic representation of proteolysis of a protein and oxidation of glycopeptides by galactose oxidase (GAO) in accordance with the present invention;
FIG. 3 is a schematic diagram showing the oxidation principle of galactose oxidase (GAO) to galactose and N-acetylgalactosamine according to the present invention;
FIG. 4 is a schematic representation of the covalent attachment of oxidized galactose and N-acetylgalactosamine glycopeptides of the present invention to hydrazide resins;
FIG. 5 is a schematic diagram of the enrichment preparation of T antigen glycopeptide immobilized enzyme of the present invention.
Detailed Description
The invention discloses a method for enriching and enzyme digestion analysis (SPTAgE) of a glycopeptide based on a solid phase glycoprotein T antigen, which comprises the following steps:
the first step is as follows: sample protein extraction
The method comprises the following steps: tissue sample protein extraction
Freezing the tissue sample in dry ice or-80 deg.C for 2-3 hours, and mashing the tissue by grinding;
adding 400-600 microliter RIPA lysate into the mashed tissue, crushing the tissue for 30 seconds by using an ultrasonic crusher (30-40% energy), cooling the sample in ice, repeating the step for 4-6 times, centrifuging at 12,000 Xg for 10 minutes, and taking the supernatant;
taking 2-4 microliter samples, diluting 5-10 times, and testing the concentration of the protein by using BCA;
according to the concentration, 800-1000 micrograms of protein is dissolved in 400-600 microliters of urea with the final concentration of 8M, and the sample is slightly shaken to ensure that the protein is completely dissolved, so that the protein solution is obtained.
The second method comprises the following steps: body fluid sample protein extraction (blood as an example)
The collected blood sample is placed in a room temperature environment to clot the sample (typically 15-30 minutes). Then, the supernatant (serum) was centrifuged at 1,000-2,000 Xg at 4 ℃ for 10 minutes. Measuring the serum protein concentration using a ultramicro-spectrophotometer;
taking 20 microliter serum sample and directly adding 180 microliter urea (the final concentration is 8M);
heat denaturation at 90 ℃ for 10 min to obtain a protein solution.
The second step is that: proteolysis
Referring to FIG. 2, FIG. 2 is a schematic diagram of the enzymatic hydrolysis of a protein and oxidation of glycopeptides by galactose oxidase (GAO) according to the present invention. As shown in FIG. 2, the protein is enzymatically hydrolyzed (trypsin) to obtain polypeptide and glycopeptide, and N-acetylgalactosamine (GalNAc) and galactose (Gal) are oxidized by GAO to form aldehyde group. Thus, the specific steps of proteolysis are as follows:
adding 1/10 volume of 120mM Dithiothreitol (DTT) to the tissue or body fluid-extracted protein solution, and reacting at 37 ℃ for 1-1.5 hours;
adding 160mM iodoacetamide with the volume of 1/10, and reacting for 0.5-1 hour in a dark room at room temperature;
diluting the sample by 5-6 times, adding newly prepared 1M ammonium bicarbonate, wherein the final ammonium bicarbonate concentration is 90-110mM, and the pH of the test sample is between 7 and 9;
adding 20-25 microgram sequencing grade trypsin, slightly oscillating, reacting at 37 deg.C for 16-18 hr, and hydrolyzing to obtain polypeptide;
adding 10-12% formic acid (w/v) to the solution until the pH is adjusted to 2-3;
pretreating the C18 extraction column, adding the sample, adding the filtrate into the extraction column, washing the extraction column with 0.1% TFA for 5-6 times (1.0-1.2 ml), eluting the polypeptide with 400-500. mu.l of 50-60% Acetonitrile (ACN) containing 0.1% TFA, and repeating the last step for 2-3 times (the proportions are volume ratios);
the washed polypeptides are combined and lyophilized in vacuo to yield the purified polypeptide.
The third step: oxidation of galactose and N-acetylgalactosamine
Referring to FIG. 3, FIG. 3 is a schematic diagram showing the oxidation principle of galactose oxidase (GAO) to galactose and N-acetylgalactosamine according to the present invention. As shown in fig. 3, 18 to 22. mu.l of dimethyl sulfoxide, 44 to 46. mu.l of sodium phosphate buffer (pH 7), 24 to 26. mu.l of horseradish peroxidase and 8 to 12. mu.l of galactose oxidase were added to the above-purified polypeptide, followed by reaction at 35 ℃ for 0.9 to 1.1 hours.
The fourth step: immobilization of hydrazide resin with PNGase F enzyme cleavage
Referring to FIG. 4, FIG. 4 is a schematic diagram illustrating the covalent binding process of oxidized galactose and N-acetylgalactosamine glycopeptides and hydrazide resin according to the present invention. As shown in FIG. 4, the glycopeptide oxidized with galactose oxidase was covalently bound to hydrazide resin, 1. mu.l of PNGase F enzyme was added, reacted at 37 ℃ for 3 hours, and then the supernatant was removed and the resin was washed.
The fifth step: galactosidase treatment and Mass Spectrometry
Please refer to FIG. 5, FIG. 5 is a schematic diagram of the enrichment preparation of T antigen glycopeptide immobilized enzyme of the present invention. As shown in fig. 5, beta galactosidase, T antigen glycopeptide and core 2 glycopeptide were enzymatically cleaved from the solid phase; core 8 glycopeptides were obtained using alpha galactosidase. Mass spectrometric detection of T antigen glycopeptides that can distinguish core 2 from core 1. The method comprises the following specific steps:
156. mu.l of deionized water, 38-42. mu.l of 50-60mM sodium phosphate buffer (pH 7-8), and 1.8-2.2. mu.l of beta-galactosidase were added to the PNGase F enzyme-treated resin, and reacted at 37 ℃ for 1 hour to cleave the linkage between galactose and acetylgalactosamine in the T antigen, thereby obtaining a polypeptide containing the T antigen (core 1) and the core 2 modification site.
And (2) analyzing the polypeptide containing the T antigen modified site obtained by enzymolysis by using liquid chromatography-mass spectrometry to obtain a primary mass spectrum and a secondary mass spectrum, analyzing mass spectrum data by using a chromatographic method of 10-50% ACN and mass spectrum energy CE 30 and bioinformatics software, wherein the T antigen modified site is increased by 203Da (GalNAc-polypeptide), and the non-site is unchanged. The core 2 glycopeptide has a GlcNAc-GalNAc-polypeptide whereas the T antigen glycopeptide is a GalNAc-polypeptide, so mass spectrometric detection of the core 2 glycopeptide has a 406Da profile.
The beta galactosidase treated resin is then treated with alpha galactosidase to cut off the linkage between galactose and acetylgalactosamine in galactose-alpha (1-3) -acetylgalactosamine (core 8 structure) and obtain the polypeptide containing core 8. Mass spectrometry and software analysis were performed under the same conditions as described above to obtain information on the site of galactose- α (1-3) -acetylgalactosamine modification, which was then distinguished from T antigen modification.
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are further described below. The invention is not limited to the embodiments shown but also encompasses any other known variations within the scope of the invention as claimed.
First, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
The present invention is described in detail by using the schematic structural diagrams, etc., and for convenience of illustration, the schematic diagrams are not enlarged partially according to the general scale when describing the embodiments of the present invention, and the schematic diagrams are only examples, which should not limit the scope of the present invention. In addition, the actual manufacturing process should include three-dimensional space of length, width and depth.
Example 1
Enrichment of proteins modified by T antigen in urine or tissue of pancreatic cancer patients versus healthy subjects
Cancer affects the O-glycosylation of specific proteins that may eventually be secreted into the urine of a patient. Therefore, the specific T antigen modified protein in the urine of pancreatic cancer patients can be identified by the invention.
(1) Urine sample collection and processing
10 ml of urine was taken from each subject and dried completely in a freeze dryer. The dried urine sample was reconstituted with 1 ml of deionized water and the protein precipitated by adding ethanol at a volume ratio of 1: 5. Then, the mixture was centrifuged at 12,000 Xg at 4 ℃ for 10 minutes, and the supernatant was collected. Urine protein concentration was determined using the Bradford method.
(2) Enzymolysis of urine protein and purification of polypeptide
Adding 300 microliters of 8M urea solution to 100 microliters of urine protein solution (10 micrograms/microliter), and performing thermal denaturation at 90 ℃ for 10 minutes;
adding 40 microliter of 120mM Dithiothreitol (DTT), and reacting for 1-1.5 hours at 37 ℃;
adding 44 microliter 160mM iodoacetamide, and reacting for 0.5-1 hour in a dark room at room temperature;
diluting the sample volume to 1.6 ml, adding 40 μ l of freshly prepared 1M ammonium bicarbonate to bring the test sample pH between 7-9;
adding 20 micrograms of sequencing-grade trypsin, slightly oscillating, reacting at 37 ℃ for 16-18 hours, and hydrolyzing to obtain polypeptide;
adding 65 microliter 10% formic acid to the solution to adjust the pH to 2-3;
pretreating the C18 extraction column (1 ml volume), adding the sample, adding the filtrate to the extraction column, washing the column with 0.1% TFA 5-6 times (1 ml each), and eluting the polypeptide 3 times with 500. mu.l of 50% Acetonitrile (ACN) containing 0.1% TFA;
the washed polypeptides are combined and lyophilized in vacuo to yield the purified polypeptide.
(3) Oxidation of galactose and N-acetylgalactosamine
To the dried polypeptide sample, 20. mu.l of dimethyl sulfoxide, 45. mu.l of sodium phosphate buffer (pH 7), 25. mu.l of horseradish peroxidase and 10. mu.l of galactose oxidase were added, followed by reaction at 35 ℃ for 1 hour
(4) Immobilization of hydrazide resin with PNGase F restriction enzyme
In the last step, glycopeptide oxidized by galactose oxidase is covalently bonded with hydrazide resin, 1 microliter of PNGase F enzyme is added, reaction is carried out at 37 ℃ for 3 hours, and then supernatant is removed and the resin is cleaned.
(5) Galactosidase treatment and Mass Spectrometry
158. mu.l of deionized water, 40. mu.l of 50-60mM sodium phosphate buffer and 2. mu.l of beta-galactosidase were added to the PNGase F enzyme-treated resin in the previous step, and reacted at 37 ℃ for 1 hour to cleave the linkage between galactose and acetylgalactosamine in the T antigen.
And (2) analyzing the polypeptide containing the T antigen modified site obtained by enzymolysis by using liquid chromatography-mass spectrometry to obtain a primary mass spectrum and a secondary mass spectrum, analyzing mass spectrum data by using a chromatographic method of 10-50% ACN and mass spectrum energy CE 30 and bioinformatics software, wherein the T antigen modified site is increased by 203Da (GalNAc-polypeptide), and the non-site is unchanged. The core 2 glycopeptide has a GlcNAc-GalNAc-polypeptide whereas the T antigen glycopeptide is a GalNAc-polypeptide, so mass spectrometric detection of the core 2 glycopeptide has a 406Da profile.
The beta galactosidase treated resin is then treated with alpha galactosidase to cut off the linkage between galactose and acetylgalactosamine in galactose-alpha (1-3) -acetylgalactosamine (core 8 structure) and obtain the polypeptide containing core 8. Mass spectrometry and software analysis were performed under the same conditions as described above to obtain site information of galactose- α (1-3) -acetylgalactosamine modification, and thus, the site information was distinguished from T antigen modification.
The method enriches the T antigen modified glycopeptide in pancreatic cancer and normal samples, adopts isotope label IBT-10 to mark the glycopeptide, and then uses MaxQuant software to analyze LC-MS/MS data, thereby achieving the quantitative analysis of the expression difference of the T antigen modified protein between pancreatic cancer patients and healthy subjects in different stages and further developing the specific glycoprotein biomarker.
Example 2
The expression of the T antigen modified protein in pancreatic cancer cells is abnormally increased compared with normal pancreatic cancer cells, so that the specific T antigen modified protein in the pancreatic cancer cells can be identified by the invention, and the influenced signal path can be researched by inhibiting the biosynthesis of the T antigen.
(1) Culture of pancreatic cells
The normal pancreatic cell line HTERT-HPNE and the two pancreatic cancer cell lines MIA PaCa-2 and AsPC-1 were combined in a 1X 104cells/cm ratio2In a 6-well plate. After the cells were attached, the medium was changed to serum-free medium and 2.5 μ M itraconazole was added to treat the cells for 48 hours.
(2) Extraction of cellular proteins
After the completion of the cell treatment, the culture medium in the 6-well plate was removed, and the cells were washed 2 times with a PBS solution. To each well was added 500 microliters of RIPA lysate (containing 50 microliters of 100 x protease inhibitor and 50 microliters of 100 x phosphatase inhibitor). Cells were collected with cell scraping in an EP tube, disrupted on ice for 30 seconds using a sonicator, cooled on ice for 30 seconds, and the operation was repeated 6 times. The EP tube was centrifuged at 12,000rpm at 4 ℃ for 15 minutes to obtain a supernatant. Protein concentration was determined using BCA method.
(3) Cell protein enzymolysis and polypeptide purification
To 100. mu.l of a cell protein solution (1. mu.g/. mu.l) was added 300. mu.l of an 8M urea solution, and heat denatured at 90 ℃ for 10 minutes;
adding 40 microliter of 120mM Dithiothreitol (DTT), and reacting at 37 ℃ for 1-1.5 hours;
adding 44 microliter 160mM iodoacetamide, and reacting for 0.5-1 hour in a dark room at room temperature;
diluting the sample volume to 1.6 ml, adding 40 μ l of freshly prepared 1M ammonium bicarbonate to bring the test sample pH between 7-9;
adding 20 micrograms of sequencing-grade trypsin, slightly oscillating, reacting at 37 ℃ for 16-18 hours, and hydrolyzing to obtain polypeptide;
adding 65 microliter 10% formic acid to the solution to adjust the pH to 2-3;
pretreating the C18 extraction column (1 ml volume), adding the sample, adding the filtrate into the extraction column, washing the extraction column with 0.1% TFA for 5-6 times (1 ml each time), and eluting polypeptide with 500 μ l of 50% Acetonitrile (ACN) containing 0.1% TFA for 3 times;
the washed polypeptides are combined and lyophilized in vacuo to yield the purified polypeptide.
(4) Oxidation of galactose and N-acetylgalactosamine
To the dried polypeptide sample, 20. mu.l of dimethyl sulfoxide, 45. mu.l of sodium phosphate buffer (pH 7), 25. mu.l of horseradish peroxidase and 10. mu.l of galactose oxidase were added, followed by reaction at 35 ℃ for 1 hour
(5) Immobilization of hydrazide resin with PNGase F restriction enzyme
In the last step, glycopeptide oxidized by galactose oxidase is covalently bonded with hydrazide resin, 1 microliter of PNGase F enzyme is added, reaction is carried out at 37 ℃ for 3 hours, and then supernatant is removed and the resin is cleaned.
(6) Galactosidase treatment and Mass Spectrometry
158. mu.l of deionized water, 40. mu.l of 50-60mM sodium phosphate buffer and 2. mu.l of beta-galactosidase were added to the PNGase F enzyme-treated resin in the previous step, and reacted at 37 ℃ for 1 hour to cleave the linkage between galactose and acetylgalactosamine in the T antigen.
And (2) analyzing the polypeptide containing the T antigen modified site obtained by enzymolysis by using liquid chromatography-mass spectrometry to obtain a primary mass spectrum and a secondary mass spectrum, analyzing mass spectrum data by using a chromatographic method of 10-50% ACN and mass spectrum energy CE 30 and bioinformatics software, wherein the T antigen modified site is increased by 203Da (GalNAc-polypeptide), and the non-site is unchanged. The core 2 glycopeptide has GlcNAc-GalNAc-polypeptide, whereas the T antigen glycopeptide is GalNAc-polypeptide, therefore mass spectrometric detection shows that the core 2 glycopeptide has 406 Da.
The beta galactosidase treated resin is then treated with alpha galactosidase to cut off the linkage between galactose and acetylgalactosamine in galactose-alpha (1-3) -acetylgalactosamine (core 8 structure) and obtain the polypeptide containing core 8. Mass spectrometry and software analysis were performed under the same conditions as described above to obtain site information of galactose- α (1-3) -acetylgalactosamine modification, and thus, the site information was distinguished from T antigen modification.
By inhibiting the biosynthesis of the T antigen in pancreatic cancer cells and analyzing the change of the T antigen modified protein and other affected proteins and signal pathways, on one hand, the understanding of mechanisms related to pancreatic cancer development can be promoted, and on the other hand, the method has important significance for finding drug treatment targets of pancreatic cancer.
In summary, compared with the prior art, the present invention provides a method based on solid phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis (SPTAgE), which comprises the steps of first, extracting protein from a biological or clinical sample by lysis. The protein is then hydrolyzed with a protease to give the polypeptide. Followed by treatment with galactose oxidase (GAO), the glycopeptide glycans are oxidized and then bound to hydrazide resins. PNGase F is then used to cleave the N-glycan to polypeptide linkage. Referring to FIG. 1, FIG. 1 is a schematic diagram of the working procedure of enriching glycopeptides containing T antigen modifications according to the present invention. As shown in fig. 1, the method yields T antigen glycopeptides of three structures, core 1: the glycopeptide of galactose-beta (1-3) -acetylgalactosamine (T antigen) is obtained by beta-galactosidase treatment; core 2 glycopeptide: galactose-beta (1-3) -acetylgalactosamine glycopeptide with acetylglucosamine is obtained by beta-galactosidase enzymolysis; core 8 glycopeptides: the glycopeptide of galactose-alpha (1-3) -acetylgalactosamine (core 8 structure) is obtained by alpha-galactosidase enzymolysis. The T antigen glycopeptide and core 2 glycopeptides are distinguished by mass spectrometry, whereas the T antigen glycopeptide is distinguished from core 8 by different glycosidases. The method can enrich and distinguish glycopeptides containing T antigen, core 2 and core 8, and has important significance in finding specific markers with T antigen modification in tumor tissues and body fluids, and research on early diagnosis and prognosis markers of cancers.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (10)
1. A method based on solid phase glycoprotein T antigen glycopeptide enrichment and enzyme digestion analysis is characterized by comprising the following steps:
(1) extracting a protein from the sample;
(2) carrying out proteolysis to obtain polypeptide;
(3) oxidation of galactose and N-acetylgalactosamine;
(4) fixing hydrazide resin and performing enzyme digestion with PNGase F;
(5) galactosidase treatment and mass spectrometry.
2. The method of claim 1, wherein in step (1), the sample comprises: tissue samples or body fluid samples.
3. The method of claim 1, wherein in step (2), the obtaining of the polypeptide by proteolysis comprises:
adding dithiothreitol into the protein solution, and reacting for 1-1.5 hours at the temperature of 37 ℃;
adding iodoacetamide, and reacting in a dark room at room temperature for 0.5-1 hour to obtain a sample;
thirdly, diluting the sample, adding ammonium bicarbonate until the final concentration of the ammonium bicarbonate is 90-110mM and the pH value of the sample is between 7 and 9;
adding sequencing-grade trypsin, slightly oscillating, and hydrolyzing at 37 ℃ for 16-18 hours to obtain a polypeptide solution;
adding formic acid into the polypeptide solution until the pH value is reduced to 2-3;
sixthly, pretreating the C18 extraction column, adding the polypeptide solution into the C18 extraction column, adding the filtrate into the C18 extraction column again, washing the C18 extraction column with 0.1% TFA for a plurality of times, finally, eluting the polypeptide with 50-60% acetonitrile containing 0.1% TFA, and repeating the steps for a plurality of times;
and seventhly, combining the eluted polypeptides, and carrying out vacuum freeze drying to obtain the purified polypeptide.
4. The method of claim 3, wherein the method comprises the steps of: in the step (3), the galactose and N-acetylgalactosamine oxidation comprises: adding dimethyl sulfoxide, sodium phosphate buffer solution, horseradish peroxidase and galactose oxidase into the purified polypeptide, and reacting at 35 ℃ for 0.9-1.1 h to obtain the glycopeptide.
5. The method of claim 4, wherein the method comprises the steps of: the volume ratio of the dimethyl sulfoxide to the sodium phosphate buffer solution to the horseradish peroxidase to the galactose oxidase is 9-11: 22-23: 12-13: 4-6.
6. The method of claim 4, wherein the method comprises the steps of: in the step (4), the hydrazide resin immobilization and PNGase F enzyme cleavage comprises: covalently bonding the glycopeptide and hydrazide resin, adding PNGase F enzyme, reacting at 37 ℃ for 3 hours, removing supernatant, and cleaning to obtain the resin.
7. The method of claim 6, wherein in step (5), the galactosidase treatment comprises:
adding deionized water, 50-60mM sodium phosphate buffer solution and beta galactosidase into the resin, and reacting for 1 hour at the temperature of 37 ℃ to cut off the connection between galactose and acetylgalactosamine in the T antigen, thereby obtaining the polypeptide containing the T antigen and the core 2 modified site.
8. The method of claim 7, wherein in step (5), the galactosidase treatment further comprises:
② adding alpha galactosidase into the resin after beta-galactosidase treatment to cut off the connection between galactose and acetylgalactosamine in galactose-alpha (1-3) -acetylgalactosamine, and obtaining the polypeptide containing core 8.
9. The method of claim 7, wherein the solid phase glycoprotein T antigen glycopeptide enrichment and cleavage assay comprises: the volume ratio of the deionized water to the sodium phosphate buffer to the beta galactosidase is 156-160: 38-42: 1.8-2.2.
10. The method of claim 8, wherein in step (5), the mass spectrometric analysis comprises: respectively analyzing the polypeptide containing the T antigen and the core 2 modification site and the polypeptide containing the core 8 by using liquid chromatography-mass spectrometry to obtain primary and secondary mass spectra, and analyzing mass spectrum data by using bioinformatics software to obtain site information.
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| WO2014145180A1 (en) * | 2013-03-15 | 2014-09-18 | Glycobia, Inc. | Polysialic acid, blood group antigens and glycoprotein expression |
| CN104198613B (en) * | 2014-09-17 | 2016-04-06 | 山东大学 | A kind of method of analyzing proteins O-glycosylation site |
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