CN109959699A - A kind of Mass Spectrometry detection method carrying out intact glycosylated peptide fragment based on quasi- multistage spectrum - Google Patents

A kind of Mass Spectrometry detection method carrying out intact glycosylated peptide fragment based on quasi- multistage spectrum Download PDF

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CN109959699A
CN109959699A CN201711339389.2A CN201711339389A CN109959699A CN 109959699 A CN109959699 A CN 109959699A CN 201711339389 A CN201711339389 A CN 201711339389A CN 109959699 A CN109959699 A CN 109959699A
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peptide fragment
deglycosylation
intact
glycopeptide
fragmentation
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CN109959699B (en
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叶明亮
秦洪强
陈尧
王璐
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Dalian Institute of Chemical Physics of CAS
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Dalian Institute of Chemical Physics of CAS
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Abstract

The present invention relates to a kind of Mass Spectrometry detection methods that intact glycosylated peptide fragment is carried out based on quasi- multistage spectrum, and its application in the early diagnosis of liver cancer.The invention mainly includes following five steps: the 1) enrichment of glycopeptide segment；2) deglycosylation peptide fragment-intact glycosylated peptide fragment sample preparation；3) deglycosylation peptide fragment-intact glycosylated peptide fragment Mass Spectrometer Method；4) deglycosylation peptide fragment-intact glycosylated peptide fragment spectral data is retrieved；5) it on this basis, in conjunction with glycosylation site, intact glycopeptide quantitative technique, realizes that the glycosylation fine difference of tumor patient and normal person are analyzed, screens potential disease markers.This method realizes the fine resolution of source of people protein glycosylation, has important application potential in terms of the screening of tumor markers.

Description

A kind of Mass Spectrometry detection method carrying out intact glycosylated peptide fragment based on quasi- multistage spectrum
Technical field
The invention belongs to glycosylated protein omics technology fields in proteomics research direction, and in particular to N- connection The application of the high throughput identification of glycopeptide segment and quantitative analysis and the technology in clinical tumor diagnosis.
Background technique
For protein glycosylation as a kind of most universal and most important posttranslational modification, it takes part in immune response, cell- The processes such as cell interaction, ligand-receptor interaction, it is abnormal closely related with the generation of disease.Currently, clinically institute Protein tumor markers are glycosylated protein mostly, pernicious swollen such as marker protein-alpha-fetoprotein (AFP) of liver cancer Marker-cancer antigen 125 (cancer antigen 125) of tumor and marker-prostate cancer specific of prostate cancer (Kyselova, Z.et al.Breast cancer diagnosis and the prognosis through such as antigen (PSA) quantitative measurements of serum glycan profiles.Clin.Chem.2008,54,1166- 1175).However, the expression quantity that current tumor markers are primarily upon its protein is horizontal, and for its modification and tumour Occur there is not yet clearly reporting, this is mainly limited by glycosylation analysis means.
In glycosylated protein group analysis, high performance liquid chromatography-mass spectrometry is scale analyzing glucoprotein/glycopeptide A kind of active platform.Since for non-glycopeptide or non-glycoprotein, the content of glycopeptide or glycoprotein is very low, and glycopeptide or The ion signal intensity of glycoprotein is often inhibited by the ion signal of non-glycopeptide or glycoprotein, and its degree of glycosylation is deposited In the microheterogeneity of height.Meanwhile the fragmentation energies of glycosidic bond are far below the fragmentation energies of peptide bond, and it is broken in turn result in glycopeptide It is difficult to obtain corresponding peptide fragment fragment information when splitting, it is difficult to realize its corresponding efficient Mass Spectrometer Method.Conventional method is using two Strategy (Woo, C.M.et al.Development of IsoTaG, a that grade spectrum-three-level spectrum (MS2-MS3) combines Chemical Glycoproteomics Technique for Profiling Intact N-and O-Glycopeptides From Whole Cell Proteomes.J.Proteome Res.2017,16,1706-1718), sugar chain knot is realized in MS2 The fragmentation of structure, and to the further MS3 fragmentation of its fragment, obtain corresponding peptide fragment skeleton fragmentation information.The method achieve sugar chains It with fragmentation while peptide fragment skeleton, however is influenced by ion transmission efficiency, while screening the ion for being used for further fragmentation It is very difficult.The above difficult point causes glycopeptide analysis throughput, sensitivity and the determination rates of MS2-MS3 strategy limited.Therefore, it realizes The accurate Analysis of glycopeptide segment still faces significant challenge.
Summary of the invention
The high throughput identification that can carry out N- connection glycopeptide segment the purpose of the present invention is to provide one kind divides with quantitative The application of analysis and the technology in clinical tumor diagnosis.
The present invention adopts the following technical scheme:
Firstly, take source of people/source of mouse tissue, the enrichment of Protein Extraction, enzymatic hydrolysis, glycopeptide segment is carried out, is obtained Then the high glycopeptide segment of relative specificity uses glucosides enzymatic treatment part intact glycopeptide, obtain and remove glycosyl accordingly Change peptide fragment, carries out mixing and corresponding chromatographic isolation with intact glycopeptide, and using the spectrum fragmentation mode of efficient step, realize Fragmentation while intact glycopeptide and deglycosylation peptide fragment, and then corresponding second level fragmentation spectrogram is obtained, and utilize corresponding spectrogram Analytic technique realizes the identification and quantification analysis of glycosylation site occupation rate and intact glycosylated peptide fragment.
Specifically comprise the steps of,
1) glycopeptide segment of enrichment is allocated as two parts, takes a copy of it that ammonium hydrogen carbonate buffer salt system is added (NH4HCO3), and a great deal of glycosidase (including PNGase F Endo F etc.), 37 DEG C of digestion 15-20h are added；
2) deglycosylation peptide fragment is re-mixed with glycopeptide segment, after dry, and is carried out together by liquid chromatogram When separate；
3) deglycosylation peptide fragment and glycopeptide segment are subjected to Fragmentation, energy selects step energy impact broken It splits, helps to realize the fragmentation efficiency of sugar chain and peptide fragment skeleton；
4) deglycosylation peptide fragment-intact glycosylated peptide fragment spectral data is retrieved, the spy generated using intact glycopeptide fragmentation Fragmentation is levied, the confirmation of sugar chain structure is carried out, and combine corresponding deglycosylation peptide fragment, confirms the peptide fragment frame sequence of intact glycopeptide Information and sugar chain structure；
5) combine glycosylation site occupation rate difference, intact glycosylated peptide fragment quantitative differences, realize tumor patient with just The glycosylation fine difference of ordinary person is analyzed, and potential disease markers are screened.
This method can obtain the qualification result of corresponding glycoprotein, glycopeptide and glycosylation site simultaneously, and in conjunction with no calibration Amount, stable isotope labeling are quantitatively used for glycosylation modified proteome analysis.
The hydrophilic enrichment material be based on click maltose material, by Dalian Chemical Physics Research Institute (Dalian, in State) teacher Liang Xinmiao laboratory synthesis epoxy nitrine maltose (5 μm).Above-mentioned glycopeptide fragmentation is the electrostatic tunnel in Thermo Road ion trap mass spectrometry is completed, equal in the instrument (mass spectrum including Velos, Q-Extractive and higher configuration) for being configured with HCD It may be implemented.The analysis software of glycopeptide is completed by the technology of this Development of Laboratories, and Java language is mainly based upon 2.0 platform of Armone.Glycosylation site occupation rate, intact glycosylated peptide fragment quantify be mainly based upon without mark label quantified At combinable stable isotope labeling is quantitatively used for its variance analysis.
Advantages of the present invention:
The method of the invention be based on deglycosylation peptide fragment-intact glycosylated peptide fragment while fragmentation, construct intend it is multiple The strategy of fragmentation spectrum, fragmentation while realizing peptide fragment frame sequence and sugar chain structure, and then realize locus specificity sugar-type Efficient identification, this method has clear advantage: advantage efficiently, high-throughput, highly sensitive, it helps to improve tumour mark The specificity of will object has important application potential in clinical diagnosis.
Detailed description of the invention
With reference to the accompanying drawing and embodiment the present invention is described in further detail:
Fig. 1 is the experiment flow figure that intact glycopeptide parsing strategy is carried out based on quasi- multiple spectral.
Fig. 2 compares for the determination rates of intact glycosylated peptide fragment under the conditions of different fragmentation energies.
Fig. 3 is to carry out intact glycopeptide based on quasi- multiple spectral to parse Policy Platform example.
Fig. 4 is that patient's cancer and cancer beside organism glycosylate variance analysis.
Specific embodiment
Following material and reagent are used in embodiment:
Acetonitrile (acetonitrile, ACN) is purchased from Yu Shan company (Shandong, China), trifluoroacetic acid (trifluoroacetic acid, TFA), formic acid (formic acid, FA) are available from Sigma company (IL, U.S.A.). Epoxy nitrine maltose (5 μm) is provided by Liang Xinmiao seminar, Dalian Chemical Physics Research Institute, (peptide glycosidase (Peptide-N- Glycosidase F, PNGase F) it is purchased from New England Biolabs company (MA, U.S.A.).Experimental water is through being purchased from The Milli-Q water treatment system of Millipore company (MA, U.S.A.) purifies.Other reagents of super filter tube are that analysis is pure or more High-purity.
Embodiment 1
This experiment mouse tissue/human liver cancer tissue used, cancer beside organism are (big by Subsidiary Second Hospital, Dalian Medical Univ. Even, China) it provides.The acquirement and use of sample are completely legal, and meet the relevant regulations of the Ethics Committee, institute.Experiment flow It is as follows:
1. the preparation of protein sample: after taking the human tissue of 50-100mg to be cleared up on ice with physiological saline, shredding, go forward side by side One step is cleaned with ice physiological saline, after in liquid nitrogen be divided into lower grind into powder, 5-10mL protein cleavage liquid is added, using ultrasound Crush method extracts albumen.Protein lysate composition are as follows: 6M guanidine hydrochloride, 50mM Tris-HCl (pH 8.0), 20mM DTT.By egg White lysate high speed centrifugation, take supernatant be added to 5 times of volumes protein pre-cooling precipitated liquid in (acetone: ethyl alcohol: acetic acid= 50:50:0.1, v/v/v), it is placed in -30 DEG C of refrigerator overnights and carries out protein precipitation.The pure acetone that albumen precipitation object is pre-chilled is washed It washs 2 times, then redissolves in final concentration 6M guanidine hydrochloride and 100mM NH4HCO3(pH=8.2) it in denaturing soln, is surveyed using BCA method Determine protein concentration.Protein solution reacts 2h opened disulfide bond with final concentration 10mM DTT at 37 DEG C, under the conditions of being protected from light It is alkylated closing with final concentration 20mM IAA reaction 30min, 100mM NH is added4HCO3Solution is by the guanidine hydrochloride of original solution Concentration dilution is 1M, is that trypsase is added in 1:20 according to enzyme and albumen quality ratio, 37 DEG C of enzymatic hydrolysis are overnight.By enzymolysis liquid through C18- Freeze-drying is enriched with to glycopeptide after SPE (Waters HLB, 60mg) desalination；
2. glycopeptide is enriched with: weighing 5mg epoxy nitrine maltose material, 200 μ l80%ACN/1%TFA, ultrasound is added 15min, 20000g are centrifuged 3min, remove supernatant, and repetition is washed 2 times, and 50 μ l 80%ACN/ are finally added in remaining precipitating 1%TFA, it is spare；Sample redissolves in 250 μ l 80%ACN/1%TFA, and 50 μ l material suspensions are added, and final volume is 300 μ L, 1200rpm vibrate 1h；Prepare 200 μ l Tip, insert 1mm C18 sieve plate, 200 μ l 80%ACN/1%TFA are added and suitably turn Speed centrifugation, loading and with supernatant at 800-1200g centrifugation wash non-specific adsorption with 200 μ l 80%ACN/1%TFA afterwards Peptide fragment, and 200 μ l30%ACN/1%FA are eluted, and are collected eluent, are divided into two parts in 1:2 ratio, are lyophilized；Take 1 part of sample It redissolves in 200 μ l 20mM NH4HCO3In, and 500Unites PNGase F is added, it is subsequently placed in 37 DEG C water bath digestions 18-20h；Obtained mixed liquor is added 2 μ l FA and terminates reaction, and intact glycopeptide is mixed with deglycosylation peptide fragment, is lyophilized；
3. glycopeptide detect: by 2 processing obtain deglycosylation peptide fragment-intact glycosylated peptide fragment sample mix after, redissolve in In 0.1%FA, LC-MS/MS analysis is carried out；Wherein, it is while improves the fragmentation of deglycosylation peptide fragment and intact glycosylated peptide fragment Efficiency, we use step fragmentation energies, i.e. the energy fragmentation mode of Stepped NCE (25 ± 5), it is made to glycosylate position The number of point and the identification number of intact glycopeptide are optimal (see Fig. 2)；
4. glycopeptide spectrum elucidation: 2.0 platform of Armone developed using early period is retrieved first with deglycosylation peptide fragment Wherein peptide fragment frame sequence, and construct corresponding glycosylation site database；It is extracted using characteristic ion (204.0875Da) The fragmentation spectrogram of intact glycopeptide, and confirm pentasaccharides nuclear structure therein, and then determine the molecular weight of sugar chain and peptide fragment；Wherein peptide Segment molecule amount is matched with deglycosylation peptide fragment, and sugar chain molecule amount is compared with data, and then determines final intact glycopeptide Structural information (see Fig. 3)；
5. source of people liver cancer and cancer beside organism glycosylate variance analysis: using the technology platform, systematic comparison patient's liver cancer Glycosylation site occupation rate, the difference of locus specificity sugar-type with cancer beside organism, screen multiple potential differential glycosylations Protein, trend is consistent with document report, has major application potentiality in clinical diagnosis (see Fig. 4).
The Mass Spectrometry detection method that intact glycosylated peptide fragment is carried out based on quasi- multistage spectrum that the present invention develops, realizes protein The fine resolution of glycosylation site occupation rate, locus specificity sugar-type has advantage efficiently, high-throughput, highly sensitive, herein On the basis of, in conjunction with glycosylation site, intact glycopeptide quantitative technique, realize tumor patient and the glycosylation fine difference point of normal person Analysis, screens potential disease markers.This method realizes the fine resolution of source of people protein glycosylation, in tumor markers Screening aspect has important application potential.

Claims (7)

1. a kind of Mass Spectrometry detection method for carrying out intact glycosylated peptide fragment based on quasi- multistage spectrum, it is characterised in that:
(1) deglycosylation peptide fragment-intact glycosylated peptide fragment sample preparation is digested using hydrophilic Interaction Chromatography rich protein sample The glycopeptide segment of enrichment is allocated as two parts by the glycopeptide segment in object, and a copy of it is taken to carry out glycosylated peptide using glycosidase The sugar chain of section simplifies processing, and the deglycosylation peptide fragment of glucosides enzymatic treatment is mixed with glycopeptide segment, while carrying out the two Detection；
(2) Mass Spectrometer Method while deglycosylation peptide fragment-intact glycosylated peptide fragment mainly utilizes mass spectrographic different fragmentation energies (comprising being not limited to step energy fragmentation mode) is realized efficiently broken while deglycosylation peptide fragment-intact glycosylated peptide fragment It splits；
(3) deglycosylation peptide fragment-intact glycosylated peptide fragment spectral data is retrieved, and the feature generated using intact glycopeptide fragmentation is broken It splits, carries out the confirmation of sugar chain structure, and combine corresponding deglycosylation peptide fragment, confirm the peptide fragment frame sequence information of intact glycopeptide And sugar chain structure.
2. according to the method described in claim 1, it is characterized by: in step (1),
1) human archeocyte or tissue sample are taken, carry out protein example extraction and enzymatic hydrolysis and glycopeptide segment enrichment；
2) sample obtained after step 1), is divided into two parts in proportion, and a copy of it is carried out at deglycosylation using glycosidase Reason, and two parts of samples are re-mixed, pending subsequent sample enrichment.
3. according to the method described in claim 1, it is characterized by: in step (2),
1) the deglycosylation peptide fragment for collecting step (1)-intact glycosylated peptide fragment mixing sample redissolves, while in a liquid phase color Spectrum is separated, and the difference of the two retention time is effectively reduced；
2) on the basis of step 1) realizes deglycosylation peptide fragment-intact glycosylated peptide fragment while separating, different matter is utilized It composes fragmentation mode (comprising being not limited to step energy fragmentation mode), carries out fragmentation while the two, obtain corresponding fragmentation Spectrogram library.
4. according to the method described in claim 1, it is characterized by: in step (3),
1) by the mass spectrum of step (2) acquisition by collecting sugared fragment ion (204.0875Da etc.) therein, confirmation is corresponding Intact glycopeptide fragmentation spectrogram, and be collected, peptide fragment frame sequence molecular weight therein is determined according to five nuclear structures of N- sugar, And obtain the molecular weight of corresponding sugar chain；
2) the direct retrieval that the remaining spectrogram of step 1) is carried out to deglycosylation peptide fragment obtains the sequence skeleton letter of corresponding peptide fragment Breath；
3) step 1) is matched with the peptide fragment molecular weight of the sugar chain information, intact glycopeptide that 2) obtain with deglycosylation peptide fragment, And control false positive rate is carried out using features such as retention time, accurate molecular weights, obtain the structural information of intact glycosylated peptide fragment.
5. according to the method described in claim 1, it is characterized by: this method can obtain simultaneously corresponding glycosylated protein, The qualification result of glycopeptide segment and glycosylation site can be used for glycosylation modified proteome analysis.
6. method according to claim 1,2,3 or 4, it is characterised in that: utilize side described in claim 1,2,3 or 4 Method respectively analyzes serum, the tissue sample of tumor patient and normal person, in conjunction with glycosylation site occupation rate difference, complete The quantitative differences of whole glycopeptide segment are realized that the glycosylation fine difference of tumor patient and normal person are analyzed, can be screened potential Disease markers.
7. according to the method described in claim 6, it is characterized by: carrying out in tumor patient and normal person's collection serum, tissue The analysis of N- connection site occupation rate, locus specificity glycopeptide realizes that it glycosylates fine structure variance analysis, obtains corresponding Differential glycosylation peptide fragment can be in malignant tumours such as liver cancer, lung cancer for improving the effect assessment of clinical diagnosis accuracy and prognosis Early diagnosis in application.
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CN115662500A (en) * 2022-10-21 2023-01-31 清华大学 Method for distinguishing structural isomers of glycan by replacing similar mass isotopes through computer simulation
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