CN109828037B - Method for high-throughput enrichment and identification of endogenous N/O-linked glycopeptides - Google Patents
Method for high-throughput enrichment and identification of endogenous N/O-linked glycopeptides Download PDFInfo
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Abstract
The invention relates to a method for enriching and detecting endogenous N-/O-linked glycopeptides based on size exclusion chromatography and hydrophilic interaction chromatography, and the method is used for early diagnosis of tumors. The invention comprises the following four steps: 1) removing high-abundance proteins, and effectively reducing the nonspecific adsorption of the proteins and removing interfering proteins by utilizing high-temperature denaturation and size exclusion effects; 2) enriching N-/O-linked glycopeptides, specifically enriching N-/O-linked glycopeptides by using hydrophilic interaction chromatography; 3) mass spectrum detection, wherein mass spectrum fragmentation energy is optimized to realize simultaneous fragmentation of a peptide fragment framework sequence and a sugar chain, and glycopeptide structure analysis is realized based on a deglycosylated peptide fragment index and a theoretical deglycosylation strategy; 4) on the basis, the glycopeptide quantification technology is utilized to realize the fine differential analysis of the glycosylation of endogenous peptide fragments in serum of a tumor patient and normal human, and potential disease markers are screened. The method realizes the fine analysis of endogenous peptide glycosylation in serum, and has important application potential for screening tumor markers.
Description
Technical Field
The invention belongs to the technical field of glycosylated proteomics in the proteomics research direction, and particularly relates to a novel method for simultaneously enriching and identifying N-and O-linked endogenous glycosylated peptide segments, and application of the technology in tumor clinical diagnosis.
Background
Endogenous peptide fragments are important biological components and markers, and structural and functional changes thereof have important effects on many diseases. The obtaining of endogenous peptide generally depends on protein denaturation, and the main methods are divided into physical methods and chemical methods, wherein the physical methods comprise high temperature, ultraviolet rays, x-rays, ultrasonic waves, high pressure, violent stirring, shaking and the like; the chemical method comprises strong acid, strong alkali, urea, guanidine salt, detergent, heavy metal salt, trichloroacetic acid, concentrated ethanol and the like. The method adopts a high-temperature denaturation method, and TFA is added to reduce the peptide fragment-protein interaction, reduce the loss of endogenous polypeptide samples, effectively remove the interference of high-abundance protein by combining the size exclusion effect of an ultrafiltration tube, obtain endogenous peptide fragments, and has simple and convenient operation and high efficiency.
Protein glycosylation, the most common and important post-translational modification, is involved in life processes such as apoptosis, immune response, cell-cell interaction, ligand-receptor interaction, and development of disease. Currently, most of the clinically used disease markers are glycosylated proteins, such as liver cancer marker protein-Alpha Fetoprotein (AFP), malignant tumor marker-cancer antigen 125(cancer antigen 125), and prostate cancer marker-prostate cancer specific antigen (PSA). Therefore, the method has very important significance for the comprehensive and intensive research on glycosylation.
In the analysis of glycosylated proteome, high performance liquid chromatography-mass spectrometry is an effective platform for analyzing glycoprotein/glycopeptide in a large scale. Because the content of glycopeptides or glycoproteins is low compared to non-glycopeptides or non-glycoproteins, the ionic signal intensity of glycopeptides or glycoproteins is often suppressed by the ionic signal of non-glycopeptides or glycoproteins. Therefore, when the protein glycosylation analysis is carried out, the pre-separation and enrichment of glycopeptide/glycoprotein are very critical.
Currently, the glycopeptide/glycoprotein enrichment methods are mainly lectin affinity chromatography, hydrophilic interaction chromatography, and hydrazide chemistry methods in which a sugar chain is covalently linked to a functional group of a material. The main principle of hydrophilic interaction chromatographic enrichment is to combine hydrophilic groups on the surface of a hydrophilic material with a large number of hydroxyl groups on a sugar chain by forming hydrophilic interaction, thereby achieving the effect of separating and enriching glycopeptides or glycoprotein. Compared with the other two enrichment methods, the method for enriching glycopeptides or glycoproteins by using hydrophilic action has the advantages of simple and convenient operation and no damage to glycoform structures, can further identify sugar chains and glycopeptides simultaneously, has more advantages, and is more suitable for analyzing complete glycosylated peptide sections.
Disclosure of Invention
The invention aims to provide an analysis method for simply, efficiently and high-flux analyzing N-/O-linked endogenous glycosylation in a complex biological sample, and the method is used for differential analysis of serum endogenous peptides of tumor patients.
The method provided by the invention is based on high-temperature denaturation to reduce the adsorption of endogenous peptide fragments, size exclusion to remove the interference of macromolecular protein and the like, and selective hydrophilic interaction chromatography is combined to realize the simultaneous enrichment and detection of N-/O-linked endogenous glycopeptides, thereby realizing the fine analysis of the glycosylation of the endogenous peptide fragments in serum.
The invention adopts the following technical scheme:
firstly, taking a biological sample, performing high-temperature denaturation and ultrafiltration to obtain an endogenous peptide segment, then enriching by clicking a maltose hydrophilic material, treating the enriched glycopeptide by using a peptide glycosidase PNGase F, and finally performing mass spectrometry on the released N-/O-linked glycopeptide to obtain the N/O-linked glycosylation sites, the N/O-linked glycopeptide and corresponding glycoprotein information in the sample.
Specifically, the method comprises the following steps of,
1) diluting a biological sample by 5-12 times of volume in 1% TFA, heating in a water bath at 90-100 ℃ for 5-10min, taking out and cooling to room temperature;
2) transferring the sample obtained after the step 1) to a 5-30kDa ultrafiltration tube, centrifuging for 20-30min at 14000g, washing twice with 1% TFA, and freeze-drying;
3) redissolving the peptide fragment obtained after the step 2) and the solution with the mass ratio of 1/10-1/3 in 80% ACN/1% TFA, and carrying out mixed oscillation incubation for more than 30min according to the mass ratio of the sample and the click maltose-water-absorbing material of 1/50-1/20;
4) adding the mixed sample obtained in the step 3) into Tip with a C18 sieve plate, centrifuging for 1-10min at a proper rotating speed in an interval of 800-2500 rpm after loading, adding 80% ACN/1% TFA with the volume 1-2 times of that of the loaded sample to wash nonspecific adsorption, finally eluting glycopeptide by using equal volume of 30% ACN/1% FA, and freeze-drying the sample;
5) putting the glycopeptide obtained in the step 4) into 50-200 microliters of 20mM NH4HCO3, adding PNGase glycosidase to the glycopeptide segment with the mass ratio of 500 units/mg-1000 units/mg, placing the glycopeptide in a water bath kettle at 37 ℃ for enzyme digestion for 18-20h, and performing enzyme digestion on the N-linked sugar chain;
6) and (3) adding FA into the mixed solution after the N-linked sugar chain is obtained in the step 5) to enable the final concentration of FA to be 1%, freeze-drying the sample, and performing LC-MS/MS analysis after 0.1% FA is redissolved to obtain the information of the N-/O-glycosylation sites, the glycopeptides and the corresponding glycoproteins in the sample.
Dispersing the material used in the step 3) by using 80% ACN/1% TFA before mixing with a biological sample, and simultaneously removing interfering hydrophilic impurities in the material, wherein the specific operations are as follows: mixing the materials and the solution at a ratio of 1mg/200 μ l-400 μ l, performing ultrasonic treatment for 10-15min, centrifuging at 20000g, repeating the above steps, and adding 80% ACN/1% TFA at a suitable ratio;
the sample and the material in the step 3) are oscillated and incubated, the material can be uniformly dispersed, and the rotating speed can be set to be 1200r/min-1300 r/min.
The centrifugation in step 4) at a suitable speed ensures that the enriched material is not drained off, so as not to reduce the enrichment efficiency.
The method can simultaneously obtain the identification results of corresponding endogenous glycoprotein, glycopeptide and glycosylation site, and can be used for proteomics analysis of glycosylation modification.
The hydrophilic enrichment based on the click maltose is carried out, and the glycopeptide enrichment material is epoxy azide maltose (5 mu m) synthesized by Liangxin 2815656by university institute of chemical and physical (Dalian, China).
The hydrophilic enrichment based on the click maltose is to enrich glycopeptides in a sample on a polypeptide level after high-temperature denaturation and ultrafiltration.
The invention has the advantages that:
the method of the invention has the obvious advantages that: simple, efficient and high-flux. Size exclusion based ultrafiltration to separate proteins and endogenous peptide fragments is widely used in proteomics analysis, however there is adsorption of proteins resulting in loss of endogenous peptide fragments. The high-temperature denatured protein can open the spatial structure of the protein, release endogenous peptide segments, enrich N-/O-linked endogenous glycopeptides through the low selectivity of a hydrophilic material, and realize the identification coverage of the N-/O-linked complete endogenous glycopeptides in serum/plasma by combining the high-resolution RPLC-MS/MS analysis and the subsequent N-/O-linked complete endogenous glycopeptide analysis technology.
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FIG. 1 Experimental flow chart of the method for enriching and detecting endogenous N-/O-linked glycosylated peptide fragments based on size exclusion chromatography and hydrophilic interaction chromatography
The invention is described in further detail below with reference to the following figures and embodiments:
Detailed Description
The following materials and reagents were used in the examples:
acetonitrile (ACN) was purchased from limonite (Shandong, China), trifluoroacetic acid (TFA), and formic acid (formic acid, FA) were purchased from Sigma (IL, U.S. A.). Epoxyazido maltose (5 μm) was provided by the university institute of chemico-physical, longstanding, title group, N281565630, (peptidoglycase F, PNGase F, available from New England Biolabs, MA, u.s.a.). experimental water was purified using a Milli-Q water treatment system available from Millipore, MA, u.s.a. other reagents in the ultrafiltration tube were analytically pure or more pure.
Example 1
The human liver cancer serum used in this experiment was provided by the second hospital (Dalian, China) affiliated to Dalian medical university. The sample was completely legal for acquisition and use and met the relevant regulations of the institutional ethics committee. The experimental procedure for the high temperature denaturing ultrafiltration and hydrophilic enrichment of serum samples was as follows:
serum (Serum) is a complex mixture containing glycoproteins, non-glycoproteins, and other substances. The endogenous N-/O-linked glycopeptide is separated and enriched by using the method based on high-temperature denaturation ultrafiltration and hydrophilic enrichment. The experimental procedure was as follows:
taking 40 μ l of Serum (60mg/ml), diluting with 1% TFA for 10 times to 400 μ l, heating in boiling water for inactivation for 5min, and cooling to 25 ℃;
2. taking a 10kDa 0.5ml ultrafiltration tube, adding 200 mu l of water, carrying out centrifugal washing at 14000g for 20min, then adding a sample, carrying out centrifugal washing at 14000g for 20min, then adding 100 mu l of 1% TFA for washing twice, and taking filtrate for freeze-drying;
3. preparation of hydrophilic chromatographic material: weighing 5mg of epoxy azide maltose material, adding 200 μ l of 80% ACN/1% TFA, performing ultrasonic treatment for 15min, centrifuging for 3min at 20000g, removing supernatant, repeating the operation and washing again, and finally adding 50 μ l of 80% ACN/1% TFA into the rest precipitate for later use;
4. redissolving the sample in 250. mu.l of 80% ACN/1% TFA, adding 50. mu.l of a suspension of hydrophilic chromatographic material to a final volume of 300. mu.l, shaking at 1200r/min for 1 h;
5. preparing 200 μ l Tip, filling into 1mm C18 sieve plate, adding 200 μ l 80% ACN/1% TFA, centrifuging at appropriate rotation speed, loading, centrifuging at appropriate rotation speed, washing non-specifically adsorbed peptide fragment with 200 μ l 80% ACN/1% TFA, eluting with 200 μ l 30% ACN/1% FA, collecting eluate, and lyophilizing;
6. re-dissolving the sample in 200 μ l of 20mM NH4HCO3, adding 500 units PNGase F, and placing the mixture in a water bath kettle at 37 ℃ for enzyme digestion for 18-20 h;
7. adding 2 mul FA into the obtained mixed solution to terminate the reaction, and freeze-drying to obtain an enriched N/O-glycopeptide sample, redissolving the enriched N/O-glycopeptide sample in 0.1 percent FA, and performing LC-MS/MS analysis.
And (3) analysis results:
as can be seen from the results in tables 1 and 2, 159 non-redundant N-glycosylation sites (corresponding to 242N-glycopeptides and 119 glycoproteins) and 523 non-redundant O-glycopeptides (corresponding to 414 glycoproteins) are identified based on the method of high-temperature denaturation ultrafiltration and hydrophilic enrichment, which indicates that the method has higher identification coverage of the N/O-glycosylation sites in serum. The method has the advantages of high-temperature denaturation ultrafiltration and hydrophilic enrichment.
And (4) conclusion: the method for enriching and detecting the endogenous N-/O-linked glycosylated peptide based on the combination of size exclusion chromatography and hydrophilic interaction chromatography, which is developed by the invention, can simultaneously realize the fine analysis of the glycosylation of the endogenous peptide in serum/plasma, has the advantages of simplicity, convenience, high efficiency and high flux, and has important application potential in the aspects of early diagnosis and prevention of tumors.
TABLE 1 identification data for O-linked endogenous glycopeptides
O-linked | Peptides | Proteins |
Sample 1 | 192 | 166 |
Sample 2 | 170 | 146 |
Sample 3 | 209 | 174 |
Total | 523 | 414 |
TABLE 2 identification data for N-linked endogenous glycopeptides
N-linked | Sites | Peptides | Proteins |
Sample 1 | 113 | 111 | 88 |
Sample 2 | 94 | 91 | 76 |
Sample 3 | 101 | 98 | 74 |
Total | 158 | 241 | 118 |
Claims (5)
1. A method for high throughput enrichment and identification of endogenous N/O-linked glycopeptides, comprising: the method comprises the following steps:
(1) removal of high abundance proteins in serum: the acting force of the protein-peptide fragment is reduced by utilizing high-temperature denaturation under an acidic condition; collecting endogenous peptide fragments by using size exclusion chromatography of an ultrafiltration membrane based on the molecular weight difference of the protein and the polypeptide;
(2) enrichment of N-/O-linked glycosylated peptide fragments: the Zip-Tip column manufactured by using improved hydrophilic interaction chromatography is added with a sample solution for centrifugation to remove non-specific adsorbed glycopeptide, and then an eluent is added for eluting a glycosylation modified peptide segment, so that a complete endogenous glycosylation peptide segment is obtained, and a part of samples are treated by PNGase F glycosidase to obtain the removed N-linked endogenous glycopeptide, thereby realizing the specific enrichment of the complete N-/O-linked endogenous glycopeptide and the deglycosylated N-linked glycopeptide;
(3) mass spectrum fragmentation and spectrogram analysis of N-/O-linked glycopeptide:
the Q-extraction high-precision mass spectrum is combined with a nano-upgrading liquid phase system, a mass spectrum fragmentation mode of data dependence and targeted fragmentation is adopted, the difference of energy fragmentation of a glycosylated peptide fragment framework and a sugar chain is utilized, and a step-based energy fragmentation mode is selected, so that simultaneous fragmentation of the peptide fragment framework and the sugar chain of N-/O-connected endogenous glycopeptides is realized, and the identification efficiency is improved;
(4) spectrogram analysis of N-/O-linked glycopeptide: combining a Mascot protein sequence search engine and a Uniprot database to realize the framework sequence retrieval of the N-/O-deglycosylated peptide segment; matching the complete glycosylated peptide segment with the deglycosylated peptide segment by utilizing an Armone glycopeptide analysis software platform based on a strategy of deglycosylated peptide segment index to realize the analysis of the N-/O-linked endogenous complete glycosylated peptide segment;
the specific method of the step (2) is as follows:
1) redissolving the endogenous peptide fragment sample collected by ultrafiltration freeze-drying in the step (1) in 80% ACN/1% TFA, adding a click maltose modified epoxy azide maltose material for enrichment, wherein the mass ratio of the sample to the hydrophilic material is 1/50-1/20, and mixing, oscillating and incubating for more than 30 min;
2) filling the mixed sample obtained in the step 1) into Tip with a C18 sieve plate, centrifuging for 1-10min, removing the sample loading solution, adding 80% ACN/1% TFA with the volume 1-2 times of that of the sample loading solution, centrifuging to remove non-specific adsorption, centrifuging to elute glycopeptide by using 30% ACN/1% FA with the same volume, collecting eluent, and directly freeze-drying the sample to obtain an endogenous glycopeptide enriched sample;
3) taking out a part of the complete endogenous glycopeptide obtained in the step 2), adding 50-200 microliters of 20mM NH4HCO3, adding PNGase F glycosidase with the mass ratio of the peptide fragment of 500 units/mg-1000 units/mg, placing in a water bath kettle at 37 ℃ for enzyme digestion for 18-20h, and releasing the N-linked sugar chain to obtain the endogenous glycopeptide without the N-linked sugar chain.
2. The method of claim 1, wherein: the specific method of the step (1) comprises the following steps:
1) diluting human serum/plasma sample with 1% TFA 5-12 times volume, placing in 90-100 deg.C water bath for 5-10min to reduce protein-polypeptide interaction;
transferring the sample obtained after the step 1) to a 5-30kDa ultrafiltration tube, centrifuging for 20-30min by 14000g, collecting filtrate, washing twice by using 1% TFA, combining the filtrates, and freeze-drying to perform subsequent sample enrichment.
3. The method of claim 1, wherein: in the step (2), the centrifugation conditions are as follows: rotating at 800-2500 rpm, and centrifuging for 1-10 min.
4. The method of claim 1, wherein: the specific method of the step (3) is as follows:
1) carrying out mass spectrum detection on the complete N-/O-linked endogenous glycopeptide collected in the step (1) by adopting an Orbitrap mass spectrum of Thermo company, wherein a secondary spectrum adopts a data-dependent mass spectrum fragmentation mode, and adopts high-low energy combined mass spectrum fragmentation to realize sugar chain fragmentation of N-linked sugar and fragmentation of an O-linked glycosylated peptide fragment skeleton; collecting the obtained N-linked deglycosylated peptide fragment, and adopting a data-dependent mass spectrum fragmentation mode, and using HCD high-energy fragmentation to obtain a sequence spectrogram of an N-linked deglycosylated peptide fragment framework;
2) the mass spectrum acquired in the step 1) is subjected to sugar fragment onium ions 204.08Da and 366.10 Da which are collected, the fragment information of O-linked sugar is confirmed, the theoretical deglycosylation strategy is utilized, the complexity of a spectrogram is effectively reduced, the skeleton sequence information of an O-linked glycosylated peptide segment is improved, and the identification of endogenous O-linked glycopeptides is realized; meanwhile, the N-linked deglycosylated peptide segment is matched with the complete glycosylated peptide segment to realize the identification of the endogenous N-linked glycopeptide.
5. Use of the method according to claim 1, characterized in that: the method can realize the fine quantitative information of the endogenous glycopeptides, including the differential analysis of glycosylation sites and site-specific sugar chains, and realize the fine differential analysis of glycosylation between tumor patients and healthy people.
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CN105467050A (en) * | 2014-09-11 | 2016-04-06 | 中国科学院大连化学物理研究所 | Identification method for O-glycosylation peptide fragment and complete saccharide chain thereof |
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CN105467050A (en) * | 2014-09-11 | 2016-04-06 | 中国科学院大连化学物理研究所 | Identification method for O-glycosylation peptide fragment and complete saccharide chain thereof |
CN106483294A (en) * | 2015-08-27 | 2017-03-08 | 中国科学院大连化学物理研究所 | A kind of selective enrichment and the method for identification N- connection glycopeptide |
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