CN111157736A - Human serum O glycosylation identification method based on chemoenzymatic - Google Patents
Human serum O glycosylation identification method based on chemoenzymatic Download PDFInfo
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Abstract
本发明涉及一种粘蛋白型O‑连接糖基化(O‑GalNAc型)检测方法。本发明基于生物酶选择性氧化‑生物正交化学反应相结合的策略，利用半乳糖氧化酶将半乳糖6‑羟基特异性氧化为醛基，并通过酰肼化学实现了糖肽的特异性捕获与释放，并利用基于质谱的检测方法实现了O‑GalNAc糖型高灵敏度分析，可以从50μL的人源血清中检测到59个O‑GalNAc型糖基化修饰序列，对应超过30个O糖化蛋白上。本方法具有检测灵敏度高、富集特异性高等特点，是对现有的内源性O‑GalNAc型糖基化蛋白/肽段分析的重要手段。
The invention relates to a method for detecting mucin-type O-linked glycosylation (O-GalNAc type). The invention is based on the combination strategy of selective oxidation of biological enzymes and biological orthogonal chemical reaction, utilizes galactose oxidase to specifically oxidize 6-hydroxyl group of galactose to aldehyde group, and realizes the specific capture of glycopeptide through hydrazide chemistry and release, and using mass spectrometry-based detection methods to achieve high-sensitivity analysis of O-GalNAc glycoforms, 59 O-GalNAc-type glycosylation modified sequences can be detected from 50 μL of human serum, corresponding to more than 30 O-glycosylated proteins superior. The method has the characteristics of high detection sensitivity and high enrichment specificity, and is an important means for the analysis of existing endogenous O-GalNAc-type glycosylated proteins/peptides.
Description
Technical Field
The invention belongs to the field of proteomics research direction O-linked glycosylated proteomics analysis, and particularly relates to a chemical enzymatic method and a liquid chromatography-mass spectrometry combined technology based enrichment and identification method for O-linked glycosylated proteins in human serum.
Background
Human serum has long been the focus of basic and clinical research because of its ability to reflect levels of human health (reference 1.Maurya, P.; Meleady, P.; Dowleng, P.; Clynes, M.Anticaner research advanced procedures for server biobased research in cancer 2007,27, 1247-. In order to be able to find more potential markers of Disease from serum, scientists looked at a number of novel methods of proteomic Analysis based on mass spectrometry (document 2.Geyer, P.E.; Kulak, N.A.; Pichler, G.; Holdt, L.M.; Teupper, D.; Man, M.cell systems Plasma protein Profiling to assessment health and Disease 2016,2,185-. N-linked glycosylated proteins are extensively studied for their high abundance in serum, but O-linked glycosylated proteins are less studied for their low abundance and micro-heterogeneity (reference 4.Chen, R., Seebun, D., Ye, M., Zou, H., Figeys, D., Site-specific characterization of cellular organization N-glycosylation with integrated hydrophilic interaction chromatography and LC-MS/MS. journal of genetics 2014,103,194 + 203.). Since abnormal levels of O-linked glycosylation are closely linked to many diseases including tumors, it is essential to develop novel and highly efficient methods for analyzing serum O-glycosylated proteome.
The currently reported analysis methods for O-glycosylated proteins in serum are limited, and most methods adopt a lectin affinity chromatography method to enrich O-linked glycosylated proteins, perform enzymolysis on the O-linked glycosylated proteins by using protease, and finally perform separation and analysis on enzymolysis products by using a liquid chromatography-mass spectrometry combined technology. Using the above method, Darula and its team identified 124O-linked glycosylation modification sites assignable to 51 proteins from 2mL of bovine serum (reference 5.Darula, Z.; Medzihradszky, K.F. molecular & cellular proteins: MCP Affinity expression and characterization of polypeptide core-1type glycosylation from bovine serum 2009,8, 2515-2526.). Recently, the team identified 27O glycosylation modification sites from at least 30 pre-fractionated fractions of human serum (reference 7.Darula, Z.; Sherman, J.; Medzihradszky, K.F. mol Cell Proteomics How to Dig Deeper. Subsequently they developed an enrichment profile using a combination of two lectins and used this method to identify 52 non-redundant O-linked glycosylation modification sites that could be assigned to 20 proteins from 400uL human serum (reference 8.Darula, Z.; Sarnyai, F.; Medzihradzky, K.F. glyco joined glycosylation sites identified from polypeptide core-1type glycoepitopes from human serum 2016,33,435-. Obviously, the identification efficiency is very low, and the requirement of large-scale screening of disease markers cannot be met, so that a novel high-sensitivity human serum O-linked glycosylation analysis method is very urgently needed.
The invention develops a chemical enzymatic method for analyzing the mucin core type 1O-linked glycosylation modification which accounts for more than 90 percent of the O-linked glycosylation level of human serum. The method comprises the steps of firstly removing N sugar chains of proteolysis peptide segments by PNGase F enzyme, then releasing sialic acid residues at the tail ends of O-linked sugar chains by an organic acid assisted method, exposing terminal galactose, then oxidizing hydroxyl groups of side chains of the proteolysis peptide segments into aldehyde groups by galactose oxidase, thereby enriching O-glycosylated protein by hydrazine groups on the surfaces of hydrazide microspheres, and finally separating and analyzing the enriched human serum O-linked glycosylation modified peptide segments by a liquid chromatography-mass spectrometry combined technology. The invention has the advantages of high enrichment specificity, high sensitivity and the like, is not interfered by residual N-linked carbohydrate chains in identification, and is an effective supplement to the existing serum O-glycosylation analysis method.
Disclosure of Invention
A great development space exists for the research of O-linked glycosylation, and finding a high-specificity enrichment means aiming at an O-linked glycosylation peptide segment is an important step for researching the O-linked glycosylation. The invention aims to develop a high-sensitivity biological sample O-linked glycosylation proteomics method based on chemoenzymatic reaction, which utilizes the principle that hydroxyl of a galactose residue side chain at the tail end of an O-linked glycosylation sugar chain is oxidized into aldehyde group by treatment of galactose oxidase, and then captures an O-linked glycosylation peptide segment through a microsphere with a hydrazide group modified on the surface by utilizing 'aldehyde-amine reaction', thereby aiming at the high-specificity enrichment and analysis of the O-linked glycosylation peptide segment.
In order to realize the purpose, the invention adopts the technical scheme that:
standard protein Fetuin treated by PNGase F glycosidase and a serum proteolysis peptide segment are taken as substrates, and the sialic acid residue is promoted to be separated from an O-linked sugar chain due to hydrolysis reaction in water bath at 75 ℃ for 1 hour in the presence of trifluoroacetic acid (TFA) by utilizing the high-temperature hydrolysis property of the sialic acid residue under the acidic condition. After removal of sialic acid residues, the terminal galactose of the O-linked carbohydrate chain was exposed, treated with galactose oxidase, the hydroxyl group of the terminal galactose side chain was oxidized to an aldehyde group, and enriched with hydrazide microspheres (as shown in fig. 1). .
The reaction comprises the following specific steps:
1) and (3) proteolysis: protein samples were dissolved in 100mM NH with 8M Urea4HCO3To the solution (pH 7.8), 20 μ L of a 1M solution of Dithiothreitol (DTT) was added, and the reaction was carried out in a water bath at 60 ℃ for 1 hour; returning to room temperature, adding 7.4mg Iodoacetamide (IAA), and reacting for 40min at room temperature in the dark; after the reaction was completed, 100mM NH was used4HCO3Solution (pH 7.8) the concentration of urea in the reaction system was diluted to a final concentration of 1M, according to enzyme: adding corresponding digestive enzyme into the protein 1:40, and performing enzymolysis overnight in water bath at 37 ℃; after the overnight reaction, the reaction was repeated according to enzyme: adding primary digestive enzyme into protein 1:40, and carrying out water bath at 37 ℃ for 6 hours; after the enzymolysis is finished, TFA is used for adjusting the pH value of a reaction system to be about 2, and Waters Oasis HLB columns are used for desalting; desalting, freeze-drying the sample, and then using 10mM NH4HCO3Redissolving the sample, adding 4. mu.L PNGase F glycosidase (500 units/. mu.L) in 37 ℃ water bath overnight, desalting with Waters Oasis HLB column after enzymolysis, collecting eluate, concentrating and drying for use.
2) Removal of sialic acid residue on O-linked sugar chain: re-dissolving the O-linked glycosylation peptide segment with the amount equivalent to 10 mu g of protein in 100 mu L of ultrapure water, carrying out vortex oscillation to uniformly disperse the peptide segment, adding 1 mu L of TFA, reacting for 1 hour at 75 ℃ after uniform mixing, and concentrating and drying the reaction system for later use after the reaction is finished.
3) Oxidation and enrichment of terminal galactose side chains: the O-linked glycosylated peptide fragment from which the terminal sialic acid residue had been removed was taken in an amount corresponding to 10. mu.g of protein, reconstituted in a half-lactose oxidation solution containing 10% DMSO (25U/mL galactose oxidase, 25mM sodium phosphate, 40U/mL horseradish peroxidase, pH 7.0) and treated overnight at 35 ℃. After the overnight treatment, the reaction system was adjusted to a pH of about 5.0 with a 50% acetic acid solution, and the reaction solution was added to hydrazide microspheres which had been washed with an oxidizing solution (100mM acetic acid, 150mM sodium chloride, pH 5.0) and bonded overnight with shaking at 25 ℃. After completion of the bonding, the hydrazide microspheres were washed three times with 50mM HEPES solution (pH 7.8), 80% ACN and 1.5M sodium chloride, respectively, with 8M urea to wash away non-specific adsorption. After completion of the washing, an elution solution (0.2M methylhydroxylamine, 0.1M sodium acetate, 1.5M sodium chloride, pH 4.5) was added and eluted overnight with shaking at 25 ℃. After elution was complete, the eluate was collected, desalted using a Waters Oasis HLB column and the sample was lyophilized for use.
4) LC-MS/MS analysis and data processing: o-linked glycosylated peptide sections with the amount equivalent to 5 mug of initial protein are re-dissolved in 7 muL of sample loading liquid (0.1 percent of FA) for full dispersion, and then the sample is injected after high-speed centrifugation. The liquid chromatography-mass spectrometry system uses an Ultimate 3000 ultra-high pressure liquid chromatography system and a Q-active mass spectrometry system of Thermo Fisher. The trap column was packed with a 5 μm particle size C using a packed column with a plug18A capillary column with an inner diameter of 200 μm filled with a self-made analytical chromatographic column with an inner diameter of 1.9 μm18A 150 μm inner diameter capillary column of packing. Mass spectrum conditions: the step normalized collision energies were 28%, 31% and 34%. The first 15 ions of each primary spectrum are taken in intensity order for secondary fragmentation. Performing database retrieval on raw data generated by mass spectrum by using Proteome scanner software, setting cysteine alkylation as fixed modification, methionine oxidation and asparagine carboxylation as variable modification, and setting enriched O-linked sugar chains as variable modification, wherein the link positions are serine/threonine residuesThe false positive rate is controlled to be less than 1 percent.
The invention has the following advantages:
1. according to the invention, the organic acid is adopted to assist the hydrolysis of sialic acid residues, so that terminal galactose is exposed, and the analysis sensitivity of the O-linked glycosylated peptide is higher.
2. The invention uses galactose oxidase to oxidize the terminal galactose of O-linked carbohydrate chain, and has high substrate oxidation specificity and high oxidation efficiency.
3. The invention enriches the O-linked glycosylated peptide section treated by galactose oxidase by using the microspheres with the surface modified with hydrazide groups, and has high enrichment specificity.
4. The invention adopts the step normalization collision energy to carry out mass spectrometry on the O-linked glycosylation peptide segment, so that the ion fragmentation of the peptide segment is more complete, the spectrogram quality is better, and the identification sensitivity is higher.
The invention relates to a method for analyzing O-linked glycosylated peptide fragments in a biological sample based on a chemical enzymatic method. The invention utilizes the characteristic that galactose oxidase can specifically oxidize side chain hydroxyl of galactose residue at the tail end of an O-linked sugar chain, firstly utilizes organic acid to assist to release sialic acid residue at the tail end of the O-linked sugar chain so as to expose the galactose residue, and then utilizes galactose oxidase to oxidize the sialic acid residue, so that the O-linked glycosylated peptide can be captured by hydrazide microspheres, thereby realizing high-specificity enrichment and high-sensitivity identification of the O-linked glycosylated peptide in a biological sample. Meanwhile, a step energy fragmentation technology is adopted in mass spectrometry, so that the identification reliability of O-linked glycosylation is improved. The invention has important application potential in the aspect of the scale analysis of O-GalNAc and other glycosylation.
Drawings
FIG. 1 Process for the enrichment of mucin-type O-glycosylation from protein hydrolysates Using a chemoenzymatic approach: (
GalNAc；
Gal；
Man；
GlcNAc ◆ NeuAc. (A) the entire course of the reaction, (B) shows a change in the glycoform structure of mucin-type O-glycosylation before and after desialyzing acid, (C) a mass increase of 27Da after covalent capture and release.
FIG. 2 hydrophilic interaction chromatography (A, C, E) and chemoenzymatic methods (B, D, F) for the target peptides: IgG enzymatic hydrolysate (untreated with PNGase F): BSA enzymatic hydrolysate 1: 2: 10 (a and B); for the target peptide: IgG enzymatic hydrolysate (treated with PNGase F): BSA enzymatic hydrolysate 1: 2: 10 enrichment effect (C and D); for the target peptide: BSA enzymatic hydrolysate 1: 10 (E and F). The molecular weight of the standard peptide after enrichment is 1240.79Da and is marked by red stars.
Figure 3 shows an annotated spectrum of the identified peptide fragment HTSVQTTSSGSGPFTDVR, which can be seen to contain reporter ions 204.0866Da and 393.1497 Da.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1
The method for analyzing the low-abundance peptide fragment in the microsystem is used for human serum proteomics analysis:
(1) dissolving human serum in buffer solution containing 8M Urea and 50mM HEPES, adding 20mM DTT with final concentration, reacting in water bath at 37 ℃ for 2h, then adding IAA with final concentration of 40mM, and reacting for 40min at 25 ℃ in a dark place;
(2) diluting the Urea concentration to 1M by using an aqueous solution of 50mM HEPES, adding tryptin with the mass ratio of 1/20 to the protein, and carrying out enzymolysis for 20h in a water bath at 37 ℃ to obtain a peptide fragment solution;
(3) adjusting the pH value of the sample obtained after the step (2) to about 2 by using TFA, and selecting corresponding C according to the mass ratio of the protein to the filler of 1/1518Removing small molecules in the solution by using a solid phase extraction column to obtain a peptide fragment eluent, and freeze-drying the peptide fragment eluent;
(4) re-dissolving the peptide fragment obtained in the step (3) in a 50mM sodium phosphate solution (pH 7.5), adding PNGase F with the mass ratio of 500Units/mg to the peptide fragment, and then placing the mixture in a water bath at 37 ℃ for enzyme digestion for 12 hours;
(5) adjusting the pH of the N-removed glycosylated peptide fragment solution obtained in the step (4) to about 2 by using TFA, and using C according to the mass ratio of the peptide fragment to the filler of 1/1518Solid phase extraction is carried out to remove small molecules in the solution, and the peptide fragment solution is obtained and freeze-dried;
(6) re-dissolving the O-linked glycosylated peptide segment obtained in the step (5) in 100 mu L of 1% TFA aqueous solution, performing vortex oscillation for 1h at 75 ℃ on a constant temperature shaking table, and performing freeze drying on a sample after the reaction is finished;
(7) redissolving the peptide fragment obtained in step (6) in a galactose oxidation solution containing 10% DMSO (25U/mL galactose oxidase, 25mM sodium phosphate, 40U/mL horseradish peroxidase, pH 7.0) and treating overnight at 35 ℃;
(8) adjusting the pH of the reaction system in the step (7) to about 5.0 by using a 50% acetic acid solution, adding the reaction solution into hydrazide microspheres which are washed by using an oxidizing solution (100mM acetic acid, 150mM sodium chloride, pH 5.0), and bonding the hydrazide microspheres overnight at 25 ℃ with shaking;
(9) the reaction system obtained in step (8) was washed three times with 50mM HEPES solution (pH 7.8) with 8M urea, 80% ACN and 1.5M sodium chloride, respectively, to wash away non-specific adsorption. After completion of the washing, an elution solution (0.2M methylhydroxylamine, 0.1M sodium acetate, 1.5M sodium chloride, pH 4.5) was added and eluted overnight with shaking at 25 ℃. After the elution is finished, collecting eluent, desalting by using a Waters Oasis HLB column, and freeze-drying a sample after desalting;
(10) performing liquid chromatography-mass spectrometry combined analysis and data processing on the peptide fragments obtained in the step (9), wherein the specific steps are as follows: the liquid chromatography-mass spectrometry system uses Ultimate 3000 ultrahigh pressure of Thermo Fisher companyLiquid chromatography system and Q-active mass system. The trap column was packed with a 5 μm particle size C using a packed column with a plug18A capillary column with an inner diameter of 200 mu m and a self-made analytical chromatographic column filled with a particle diameter C of 1.9 mu m18A 150 μm inner diameter capillary column of packing. Mass spectrum conditions: the step normalized collision energies were 28%, 31% and 34%. The first 15 ions of each primary spectrum are taken in intensity order for secondary fragmentation. And performing database retrieval on raw data generated by mass spectrum by using protome scanner software, setting cysteine alkylation as fixed modification, setting methionine oxidation and asparagine carboxylation as variable modification, taking enriched O-linked sugar chains as variable modification, setting the link positions as serine/threonine residues, and controlling the false positive rate to be less than 1%.
In conclusion, the novel method for efficiently identifying the O-linked glycosylated peptide segments in different samples combines a plurality of enzymatic reactions with reactions for releasing sialic acid by a chemical method, and can greatly improve the enrichment specificity, the identification sensitivity and the reliability of the O-linked glycosylated peptide segments. The method has wide application range and simple operation, and can be used for analyzing O-glycosylation proteomics aiming at different types of samples.
Claims (9)
1. A human serum O glycosylation identification method based on chemical enzymatic catalysis is a mucin type O-linked glycosylation (O-GalNAc type) detection method, and is characterized in that: the terminal sugar unit is specifically oxidized by using the oxidase, and O-linked glycosylation capture-release is carried out by using a hydrazide chemical method, so that the specific enrichment and detection of O-GalNAc type glycosylation are realized.
2. The method of claim 1, wherein: enriching O-GalNAc type glycoprotein/peptide segment, oxidizing hydroxyl of terminal galactose side chain into aldehyde group by using galactose oxidase, and capturing glycopeptide/protein by chemical reaction between aldehyde group and amino group.
3. The method according to claim 1 or 2, characterized in that: a pretreatment process for O-GalNAc type glycoprotein/peptide fragment enrichment, wherein a galactose/acetylgalactosamine is exposed to the end of an O-linked sugar chain for subsequent oxidation and enrichment by hydrolyzing the terminal sialic acid residue of the sugar chain under acidic conditions (pH 2) using a high temperature to release the terminal sialic acid residue of the glycopeptide.
4. The method according to claim 1 or 2, characterized in that: the enrichment and oxidation process of O-GalNAc type glucoprotein/peptide segment utilizes galactose oxidase to treat overnight, and the alcohol hydroxyl group of 6-terminal galactose/acetylgalactosamine side chain is specifically oxidized into aldehyde group.
5. The method according to claim 1 or 2, characterized in that: enriching the O-GalNAc type glycoprotein/peptide segment, and capturing the galactose/acetylgalactosamine modified peptide segment oxidized by aldehyde group by using a hydrazide chemical strategy and using microspheres modified with hydrazide groups on the surfaces through chemical reaction between amine groups and aldehyde groups; the O-methylhydroxylamine is utilized to release the galactose/acetyl galactose modified peptide segment, thereby realizing the specific enrichment of the O-GalNAc type glycoprotein/peptide segment.
6. The method of claim 1, wherein: and (3) performing mass spectrum detection on the O-GalNAc type glycoprotein/peptide fragment, performing mass spectrum fragmentation on the enriched O-GalNAc type glycosylated peptide fragment sample, and performing fragmentation by using step energy, so that the fragmentation efficiency of the sugar chain and the peptide fragment framework sequence is improved, and the identification reliability of the O-linked glycosylated peptide fragment is further improved.
7. The method according to claim 1 or 6, characterized in that: through O-GalNAc type glycoprotein/peptide fragment detection spectrogram analysis, after sialic acid residues are released and subjected to hydrazide enrichment, the number of candidate retrieval glycoform structures is reduced, the library search space and the random matching error rate are obviously reduced, direct library search can be performed, the variable modification comprises that 230.0903Da, 212.0797Da, 392.1431Da and 374.1325Da are respectively added to serine (Ser) and threonine (Thr), corresponding neutral loss is set, and the identification efficiency of the O-linked glycosylation peptide fragment is improved.
8. The method according to any one of claims 1 to 7, wherein: the terminal sugar unit refers to the outermost monosaccharide unit of the sugar chain modified on the O-GalNAc type glycoprotein/peptide fragment, part of the proteins in the raw material belong to the O-GalNAc type glycoprotein, and the glycoproteins all comprise the terminal sugar unit.
9. The method according to any one of claims 1 to 8, wherein: the research object of the strategy is the endogenous O-linked glycosylation analysis in the tissues or cells of mammals, the adopted chemical reaction reagents are all common reagents, the reaction conditions are mild, the side reaction efficiency is low, the detection sensitivity of glycopeptide can be obviously improved, and the strategy can be used for O-linked glycosylation proteome analysis of various biological samples.
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