CN109959699B - Mass spectrum detection method for complete glycosylated peptide segment based on quasi-multistage spectrum - Google Patents
Mass spectrum detection method for complete glycosylated peptide segment based on quasi-multistage spectrum Download PDFInfo
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Abstract
The invention relates to a mass spectrometry detection method for a complete glycosylation peptide section based on quasi-multistage spectroscopy and application thereof in early diagnosis of liver cancer. The invention mainly comprises the following five steps: 1) enrichment of glycosylated peptide fragments; 2) preparing a sample of deglycosylated peptide fragment-complete glycosylated peptide fragment; 3) mass spectrum detection of the deglycosylated peptide fragment-complete glycosylated peptide fragment; 4) searching spectrogram data of the deglycosylated peptide fragment-complete glycosylated peptide fragment; 5) on the basis, the glycosylation site and the complete glycopeptide quantification technology are combined, so that the glycosylation fine difference analysis of a tumor patient and a normal person is realized, and potential disease markers are screened. The method realizes the fine analysis of human protein glycosylation, and has important application potential in the screening aspect of tumor markers.
Description
Technical Field
The invention belongs to the technical field of glycosylated proteomics in the proteomics research direction, and particularly relates to high-throughput identification and quantitative analysis of an N-linked glycosylated peptide fragment and application of the technology in tumor clinical diagnosis.
Background
Protein glycosylation, the most common and important post-translational modification, is involved in immune responses, cell-cell interactions, ligand-receptor interactions, etc., and its abnormalities are closely related to the occurrence of disease. Currently, the tumor markers of the clinically used proteins are mostly 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) (kysleova, z.et al. However, the current tumor markers mainly focus on the expression level of the protein, and the modification and tumor occurrence are not clearly reported, which is mainly limited by glycosylation analysis means.
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, and the glycosylation degree is highly microscopically heterogeneous. Meanwhile, the fragmentation energy of the glycosidic bond is far lower than that of the peptide bond, so that corresponding peptide fragment information is difficult to obtain during glycopeptide fragmentation, and corresponding high-efficiency mass spectrum detection is difficult to realize. The conventional method adopts a strategy of combining secondary spectrum-tertiary spectrum (MS2-MS3) (Woo, C.M. et al. development of IsoTaG, a Chemical glycobiology techniques for Profiling introduced N-and O-glycoepides from Cell proteins. J.protein Res.2017,16,1706-1718), realizes the fragmentation of sugar chain structure in MS2, and further performs MS3 fragmentation on the fragments to obtain corresponding peptide fragment framework fragmentation information. This method realizes simultaneous fragmentation of sugar chain and peptide fragment frameworks, however, is affected by ion transport efficiency, and it is very difficult to screen ions for further fragmentation at the same time. The above difficulties lead to limitations in glycopeptide analysis throughput, sensitivity and identification efficiency of the strategy MS2-MS 3. Thus, achieving accurate resolution of glycosylated peptide fragments still faces significant challenges.
Disclosure of Invention
The invention aims to provide a method for carrying out high-throughput identification and quantitative analysis on N-linked glycosylated peptide fragments and application of the technology in clinical diagnosis of tumors.
The invention adopts the following technical scheme:
firstly, human source/mouse source tissues are taken to carry out the steps of protein extraction, enzymolysis, enrichment of glycosylated peptide segments and the like, the glycosylated peptide segments with high relative specificity are obtained, then glycosidase is adopted to treat partial complete glycopeptides in the glycosylated peptide segments to obtain corresponding deglycosylated peptide segments, the deglycosylated peptide segments are mixed with the complete glycopeptides and are subjected to corresponding chromatographic separation, an efficient step type spectrum fragmentation mode is adopted, the complete glycopeptides and the deglycosylated peptide segments are simultaneously fragmented, a corresponding secondary fragmentation spectrogram is obtained, and the identification and quantitative analysis of the glycosylation site occupancy rate and the complete glycosylated peptide segments are realized by utilizing a corresponding spectrogram analysis technology.
Specifically, the method comprises the following steps of,
1) dividing the enriched glycosylated peptide segment into two parts, taking one part, adding ammonium bicarbonate buffer salt system (NH)4HCO3) Adding a considerable amount of glycosidase (including PNGase F \ Endo F and the like), and carrying out enzyme digestion at 37 ℃ for 15-20 h;
2) remixing the deglycosylated peptide fragments and the glycosylated peptide fragments, drying, and simultaneously separating by liquid chromatography;
3) mass spectrum fragmentation is carried out on the deglycosylated peptide fragments and the glycosylated peptide fragments, and the energy of the deglycosylated peptide fragments and the glycosylated peptide fragments is selected to be step-type energy collision fragmentation, so that the fragmentation efficiency of sugar chains and peptide fragment frameworks is realized;
4) searching spectrogram data of the deglycosylated peptide fragment-complete glycosylated peptide fragment, confirming a sugar chain structure by utilizing characteristic fragmentation generated by complete glycopeptide fragmentation, and confirming peptide fragment framework sequence information and the sugar chain structure of the complete glycopeptide by combining with the corresponding deglycosylated peptide fragment;
5) and the glycosylation fine difference analysis of the tumor patient and the normal person is realized by combining the glycosylation site occupancy difference and the quantitative difference of the complete glycosylation peptide segment, and potential disease markers are screened.
The method can simultaneously obtain the identification results of corresponding glycoprotein, glycopeptide and glycosylation sites, and is combined with non-standard quantity and stable isotope labeling quantity for proteomics analysis of glycosylation modification.
The hydrophilic enrichment material is based on click maltose material, epoxy azide maltose (5 μm) synthesized by jongxin 2815639 of institute of chemico-physical of university (da lian, china). The glycopeptide fragmentation is completed by Thermo electrostatic tunnel ion trap mass spectrometry, and can be realized by instruments (including Velos, Q-extraction and higher mass spectrometry) provided with HCD. Glycopeptide analysis software is implemented by the technology developed in the laboratory, and is mainly based on the Java language Armone 2.0 platform. The glycosylation site occupancy rate and the quantification of the complete glycosylation peptide segment are mainly completed on the basis of label-free quantification, and can be combined with stable isotope labeling quantification for differential analysis.
The invention has the advantages that:
the method constructs a strategy of quasi-multiple fragmentation spectrum based on simultaneous fragmentation of deglycosylated peptide fragments-complete glycosylated peptide fragments, realizes simultaneous fragmentation of a peptide fragment framework sequence and a sugar chain structure, and further realizes efficient identification of site-specific glycoforms, and has the obvious advantages of: high efficiency, high flux and high sensitivity, is beneficial to improving the specificity of the tumor marker, and has important application potential in clinical diagnosis.
Drawings
The invention is described in further detail below with reference to the following figures and embodiments:
FIG. 1 is a flow chart of the experiment for performing the strategy of analyzing a complete glycopeptide based on quasi-multispectral analysis.
FIG. 2 is a comparison of the efficiency of identification of intact glycosylated peptide fragments under different fragmentation energies.
FIG. 3 is an example of a strategy platform for performing complete glycopeptide analysis based on quasi-multispectral analysis.
FIG. 4 is the analysis of the glycosylation differences between the cancer and the tissue beside the cancer.
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
Mouse tissue/human liver cancer tissue and tissues beside cancer used in the experiment were 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 was as follows:
1. preparation of protein samples: cleaning human tissue 50-100mg on ice with normal saline, cutting, further cleaning with ice normal saline, grinding into powder under liquid nitrogen, adding 5-10mL protein lysate, and treating with ultrasoundAnd (3) extracting protein by a crushing method. The protein lysate consists of: 6M guanidine hydrochloride, 50mM Tris-HCl (pH 8.0), 20mM DTT. The protein lysate was centrifuged at high speed, the supernatant was added to 5 volumes of protein pre-cooled pellet (acetone: ethanol: acetic acid: 50:0.1, v/v/v), and the mixture was placed in a freezer at-30 ℃ overnight for protein precipitation. The protein precipitate was washed 2 times with pre-cooled pure acetone and then redissolved in 6M guanidine hydrochloride and 100mM NH at the final concentration4HCO3Protein concentration was measured by BCA method in a denaturing solution (pH 8.2). The protein solution is reacted with DTT with a final concentration of 10mM for 2h at 37 ℃ to open disulfide bonds, and then reacted with IAA with a final concentration of 20mM for 30min under the condition of keeping out of the light to carry out alkylation blocking, and 100mM NH is added4HCO3The solution is prepared by diluting the original solution with guanidine hydrochloride concentration to 1M, adding trypsin according to the enzyme-protein mass ratio of 1:20, and performing enzymolysis at 37 ℃ overnight. Desalting the enzymolysis solution with C18-SPE (Waters HLB,60mg), and lyophilizing to obtain glycopeptide for enrichment;
2. and (3) glycopeptide enrichment: weighing 5mg of epoxy azide maltose material, adding 200 μ l of 80% ACN/1% TFA, performing ultrasonic treatment for 15min, centrifuging at 20000g for 3min, removing supernatant, washing repeatedly for 2 times, and adding 50 μ l of 80% ACN/1% TFA into the rest precipitate; the sample was reconstituted in 250. mu.l 80% ACN/1% TFA, 50. mu.l of the material suspension was added to a final volume of 300. mu.l, and shaken at 1200rpm for 1 h; preparing 200 μ l Tip, filling into 1mm C18 sieve plate, adding 200 μ l 80% ACN/1% TFA, centrifuging at appropriate speed, loading and centrifuging with 800-1200g supernatant, washing the 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 mixing according to 1:2, dividing into two parts according to the proportion, and freeze-drying; 1 sample was reconstituted in 200. mu.l 20mM NH4HCO3Adding 500 units PNGase F, and then placing in a water bath kettle at 37 ℃ for enzyme digestion for 18-20 h; adding 2 mul FA into the obtained mixed solution to terminate the reaction, mixing the complete glycopeptide and the deglycosylated peptide fragment, and freeze-drying;
3. and (3) glycopeptide detection: mixing the deglycosylated peptide fragment-complete glycosylated peptide fragment samples obtained by the treatment of the step 2, redissolving the mixture in 0.1% of FA, and performing LC-MS/MS analysis; wherein, in order to improve the fragmentation efficiency of deglycosylated peptide fragments and intact glycosylated peptide fragments, step fragmentation energy, namely the energy fragmentation pattern of Stepped NCE (25 +/-5), is adopted, so that the number of glycosylation sites and the identification number of intact glycopeptides are optimal (see figure 2);
4. glycopeptide spectrogram analysis: firstly, searching a peptide fragment framework sequence by using a deglycosylated peptide fragment and constructing a corresponding glycosylation site database by using an early-developed Armone 2.0 platform; extracting fragmentation spectrogram of the complete glycopeptide by using the characteristic onium ions (204.0875Da), and confirming a pentasaccharide core structure in the fragmentation spectrogram, thereby determining the molecular weight of a sugar chain and a peptide fragment; wherein the molecular weight of the peptide segment is matched with the deglycosylated peptide segment, and the molecular weight of the sugar chain is compared with the data, thereby determining the structural information of the final complete glycopeptide (see fig. 3);
5. glycosylation difference analysis of human liver cancer and tissues beside the cancer: by adopting the technical platform, the glycosylation site occupancy rate and the site-specific glycoform difference of the liver cancer and the tissues beside the cancer of the patient are compared systematically, a plurality of potential differential glycosylation proteins are screened, the trend of the potential differential glycosylation proteins is consistent with that reported in the literature, and the potential for significant application in clinical diagnosis is provided (see figure 4).
The mass spectrum detection method for the complete glycosylated peptide section based on the quasi-multilevel spectrum realizes the fine analysis of the protein glycosylation site occupancy rate and site specificity glycoform, has the advantages of high efficiency, high flux and high sensitivity, and realizes the glycosylation fine difference analysis of tumor patients and normal people and the screening of potential disease markers by combining the glycosylation site and the complete glycopeptide quantitative technology on the basis. The method realizes the fine analysis of human protein glycosylation, and has important application potential in the screening aspect of tumor markers.
Claims (6)
1. A mass spectrum detection method for complete glycosylated peptide segments based on quasi-multistage spectroscopy is characterized by comprising the following steps:
(1) preparing a sample of deglycosylated peptide fragments-complete glycosylated peptide fragments, enriching the glycosylated peptide fragments in protein sample zymolyte by using hydrophilic interaction chromatography, dividing the enriched glycosylated peptide fragments into two parts, taking one part of the two parts, adopting glycosidase to simplify sugar chains of the glycosylated peptide fragments, mixing the deglycosylated peptide fragments treated by the glycosidase with the glycosylated peptide fragments, and simultaneously detecting the two parts;
(2) the simultaneous mass spectrometric detection of the deglycosylated peptide fragment-complete glycosylated peptide fragment mainly utilizes different fragmentation energies (including but not limited to a step energy fragmentation mode) of a mass spectrum to realize the simultaneous efficient fragmentation of the deglycosylated peptide fragment-complete glycosylated peptide fragment;
(3) searching spectrogram data of the deglycosylated peptide fragment-complete glycosylated peptide fragment, confirming a sugar chain structure by utilizing characteristic fragmentation generated by complete glycopeptide fragmentation, and confirming peptide fragment framework sequence information and the sugar chain structure of the complete glycopeptide by combining with the corresponding deglycosylated peptide fragment;
in the step (1), the step (c),
1) taking a human cell or tissue sample, and extracting and carrying out enzymolysis on a protein sample and enriching a glycosylated peptide section;
2) dividing the sample obtained after the step 1) into two parts according to a proportion, wherein one part is deglycosylated by glycosidase, and mixing the two parts of the sample again to be subjected to subsequent sample enrichment.
2. The method of claim 1, wherein: in the step (2),
1) redissolving the deglycosylated peptide fragment-complete glycosylated peptide fragment mixed sample collected in the step (1), and simultaneously separating by primary liquid chromatography, thereby effectively reducing the difference of retention time of the deglycosylated peptide fragment and the complete glycosylated peptide fragment;
2) on the basis of realizing simultaneous separation of the deglycosylated peptide fragment and the complete glycosylated peptide fragment in the step 1), simultaneous fragmentation of the deglycosylated peptide fragment and the complete glycosylated peptide fragment is carried out by utilizing different mass spectrum fragmentation modes (including but not limited to a step energy fragmentation mode), so as to obtain a corresponding fragmentation spectrum library.
3. The method of claim 1, wherein: in the step (3), the step (c),
1) collecting sugar fragment onium ions (204.0875Da and the like) in the mass spectrum collected in the step (2), confirming a corresponding complete glycopeptide fragmentation spectrogram, collecting, determining the molecular weight of a peptide fragment framework sequence according to the five-core structure of the N-sugar, and obtaining the molecular weight of a corresponding sugar chain;
2) directly searching deglycosylated peptide fragments of the rest spectrograms in the step 1) to obtain sequence skeleton information of the corresponding peptide fragments;
3) matching the sugar chain information obtained in the steps 1) and 2), the molecular weight of the peptide segment of the complete glycopeptide with the deglycosylated peptide segment, and controlling the false positive rate by using the characteristics of retention time, accurate molecular weight and the like to obtain the structural information of the complete glycosylated peptide segment.
4. The method of claim 1, wherein: the method can simultaneously obtain the identification results of corresponding glycosylated proteins, glycosylated peptide sections and glycosylation sites, and can be used for proteomic analysis of glycosylation modification.
5. A method according to claim 1, 2 or 3, characterized in that: the method of claim 1, 2 or 3 is used for analyzing serum and tissue samples of tumor patients and normal people respectively, and glycosylation fine difference analysis of the tumor patients and the normal people is realized by combining glycosylation site occupancy difference and quantitative difference of complete glycosylation peptide fragments, so that potential disease markers can be screened.
6. The method of claim 5, wherein: the analysis of N-connection site occupancy and site specificity glycopeptide in serum and tissue collected by tumor patients and normal people is carried out, the glycosylation fine structure differential analysis is realized, and the corresponding differential glycosylation peptide segment is obtained, is used for improving the clinical diagnosis accuracy and the prognosis effect evaluation, and can be applied to the early diagnosis of malignant tumors such as liver cancer, lung cancer and the like.
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