CN105445358B - Analysis method based on mass spectrographic oxygen connection nitrogen acetylglucosamine modified glucoprotein matter - Google Patents
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Abstract
The present invention relates to one kind to connect nitrogen acetylglucosamine (O-GlcNAc) modified glucoprotein matter analysis method based on mass spectrographic oxygen.Peptide fragment is generated after the O-GlcNAc modified glucoprotein matter protease hydrolyzed, utilize sodium periodate oxidation, contraposition hydroxyl on modification group nitrogen acetylglucosamine carries out ring-opening reaction and forms two aldehyde groups, using on girard reagent T (Girard Reagent T) acyl trap and newly-generated aldehyde radical react, to make quaternary ammonium salt group in the chemical derivatization of O-GlcNAc modification glycopeptide.The presence of quaternary ammonium salt group can effectively enhance the mass spectrum response signal of peptide fragment, and the glycopeptide after deriving is retaining certain raw sugar modification group, subsequent mass spectrum is carried out, this kind of glycosyl modified glycoprotein is analyzed and identified by analysis means such as database search.
Description
Technical field
One kind connecting nitrogen acetylglucosamine (O-GlcNAc) modified glucoprotein matter analysis method based on mass spectrographic oxygen.Described
Peptide fragment is generated after O-GlcNAc modified glucoprotein matter protease hydrolyzed, utilizes sodium periodate oxidation, modification group nitrogen Acetylglucos
Contraposition hydroxyl on amine carries out ring-opening reaction and forms two aldehyde groups, utilizes girard reagent T (Girard Reagent T)
On acyl trap and the reaction of newly-generated aldehyde radical, to make quaternary ammonium salt group in the chemical derivatization of O-GlcNAc modification glycopeptide.Quaternary ammonium
The presence of salt groups can effectively enhance the mass spectrum response signal of peptide fragment, and the glycopeptide after deriving is repaired retaining certain raw sugar
Group is adornd, subsequent mass spectrum is carried out, this kind of glycosyl modified glycoprotein is analyzed by analysis means such as database search
Identification.
Background technique
Amine-modified oxygen connection nitrogen Acetylglucos are a kind of occur after the translation on protein serine and threonine residues
Modification is monosaccharide posttranslational modification important in a kind of eukaryocyte, plays the part of important function in signal path in the cell.So
And there are many difficult points for the glycoprotein glycoprotein Quality Research of this kind of posttranslational modification: abundance is low, glycopeptide accounts for total peptide fragment ratio
Small, mass signal response is low, not by the specific reagent etc. of modification group excision.
Mainly there are the derivative concentration method of enzymatic and agglutinin parent to the common research method of this kind of glycoprotein in document report
And method.In addition document (Journal of Proteome Research 2010,9,2200-2206) reports one kind recently
Aldehyde radical is formed using sodium periodate oxidation nitrogen acetylglucosamine, the work of this kind of glycoprotein of research is enriched with using acyl trap gel, it should
Work develops the means using chemical means research nitrogen acetylglucosamine modified glucoprotein matter, the derivative hand of enzymatic mutually costly
Section, provides possibility for cheap broad scale research nitrogen acetylglucosamine modified glucoprotein matter.
The modification of oxygen connection nitrogen acetylglucosamine (O-GlcNAc) can inhibit the mass spectrum response signal of albumen and peptide, therefore increase
The mass spectrum response signal of the strong glycopeptide, which is based on mass spectrographic analyze and identify to it, will great help.Spread out on protein or peptide fragment
Quaternary ammonium salt group changes sample charge etc. and has a wide range of applications in enhancing sample mass signal in life.Therefore, Wo Mentong
The modification for crossing quaternary ammonium salt group can effectively improve mass signal, provide help for subsequent mass spectral analysis research.
Oxygen connects in the second order ms fragmentation of glycopeptide, and under CID fragmentation mode, collision fragmentation energies often concentrate on sugar chain
Fragmentation, preferable peptide fragment fragmentation information can not be obtained.In this method, Ji is being carried out to the nitrogen Acetylglucos amine groups of modification
The chemical derivatization of ladd reagent T, the peptide fragment for modifying it can obtain preferable fragment information under CID fragmentation mode.Simultaneously
This method also remains the structural information of part nitrogen Acetylglucos amine groups, can have using the characteristic peak ion of second order ms spectrogram
Effect improves the confidence level of spectrum analysis.
Summary of the invention
Quaternary ammonium salt group can enhance sample mass signal in derivative, while the chemical derivatization is conducive to peptide fragment and obtains preferably
CID second level fragmentation, obtain good peptide fragment qualification result for follow-up data library searching and help be provided.The purpose of the present invention is mention
Pass through for a kind of pair of oxygen connection nitrogen acetylglucosamine modified glucoprotein matter and the mass spectrometric analysis method of glycopeptide, this method to the glycosyl
The chemical derivatization for changing modification improves glycopeptide mass signal, and can obtain good second order ms fragment information.Make subsequent
Database search can preferably identify that oxygen connects nitrogen acetylglucosamine modified glucoprotein matter.
To achieve the above object, the technical solution adopted by the present invention are as follows:
Peptide fragment is generated after O-GlcNAc modified glucoprotein matter protease hydrolyzed, utilizes sodium periodate oxidation, modification group nitrogen
Contraposition hydroxyl on acetylglucosamine carries out ring-opening reaction and forms two aldehyde groups, utilizes girard reagent T (Girard
Reagent T) on acyl trap and the reaction of newly-generated aldehyde radical, to make quaternary ammonium salt in the chemical derivatization of O-GlcNAc modification glycopeptide
Group.The presence of quaternary ammonium salt group can effectively enhance the mass spectrum response signal of peptide fragment, and the glycopeptide after deriving is retaining
Certain raw sugar modification group, carries out subsequent mass spectrum, by analysis means such as database search to this kind of glycosyl modified glycoprotein
Matter is analyzed and identified.
The oxygen connects nitrogen acetylglucosamine modified glucoprotein matter mass spectrometric analysis method, the specific steps are as follows:
(1) enzymatic hydrolysis of protein example: digesting after carrying out denaturation reductive alkylation to protein example, obtains conduct
The peptide fragment of enzymolysis product.3-6 μ l 1M dithiothreitol (DTT) (DTT) is added in every 100 μ g protein, 50-60 DEG C reaction 1-2 hours.It
8-12 μ l 1M iodoacetic acid (IAA) is added afterwards, is protected from light 10-30 minutes.By treated, protein example utilizes ammonium hydrogen carbonate
Buffer dilution, pH value control between 7.5-8.5, and every 100 μ g protein is added 2-5 μ g trypsase and is digested, and 36-38 DEG C
Reaction 16-24 hours.Peptide hydrolysis removes the small molecule salts such as ammonium hydrogen carbonate using reversed-phase liquid chromatography, obtains peptide after freeze-drying
Section sample carries out the peptide fragment sample of acquisition for side reaction existing when to avoid peptide fragment N-terminal be serine or threonine residues
Formaldehyde di-methylation method closes N-terminal amino.3-5% formalin 10-25 μ l, 0.5-0.8mM cyanogen is added in every 100 μ g peptide fragment
Base sodium borohydride 10-25 μ l reacts 0.5-2 hours, and reverse phase desalination removes formaldehyde and sodium cyanoborohydride.
(2) nitrogen acetylglucosamine radical oxidation: in the 50mM sodium acetate buffer solution of pH=5-6, oxygen connects nitrogen acetyl
The excess sodium periodate solution reaction that aminoglucose modified glucoprotein matter or glycopeptide sample and concentration are 20-30mM；Reaction temperature 35-
40 DEG C, reaction time 4-8 hour.After oxidation reaction, for excessive sodium metaperiodate, 4-6 times of sodium metaperiodate is added and rubs
You measure sodium sulfite, react 10-40 minutes, terminate oxidation reaction.
(3) girard reagent derivatization reaction: by by sodium periodate oxidation glycoprotein sample in concentration be 10-
The girard reagent of 100mM mixes, and reacts 1-6 hours.Reaction process carries out in the sodium acetate solution of pH=5-6.
(4) unreacted girard reagent is separated off using reversed-phase liquid chromatography: is lived using reverse phase C18 or C8 chromatography, flowed
Dynamic phase A:2% acetonitrile, 98% water and 0.1% trifluoroacetic acid.Mobile phase B: 98% acetonitrile, 2% water and 0.1% trifluoroacetic acid.It adopts
With 2% Mobile phase B loading, rinsed chromatographic column 5-15 minutes with sample-loading buffer later, it later will be sugared with 80% mobile phase
Protein or glycopeptide sample elution get off, freeze-drying.
(5) LC-MS is used, sample analysis is carried out using mass spectrum after separating to peptide fragment: utilizing collision induced dissociation
(Collision-induced dissociation, CID) mode carries out second level fragmentation to peptide fragment, for ion trap mass spectrometry, CID
Energy hole is in 30-50%.Data are acquired to analyze using the use of information that proteome databases search engine carries out glycopeptide.
(6) acquisition data are analyzed using the use of information that proteome databases search engine carries out glycopeptide: database is searched
It is amine-modified for oxygen connection nitrogen Acetylglucos in the parameter setting of rope, increase the setting of variable modification: 1) decorating molecule formula
C13H22N4O5, decorating site serine and threonine residues.2) decorating molecule formula C18H33N7O5, decorating site serine and Soviet Union ammonia
Sour residue.It is modified for di-methylation, increases variable modification: 1) decorating molecule formula C2H4, modify position lysine residue.2) it modifies
Molecular formula C2H4, decorating site peptide fragment nitrogen end.It searches library result to be further processed, for the confidence level for increasing result, utilizes second level matter
Characteristic peak in spectrogram is further screened.For having modified the modification peptide fragment of a molecule girard reagent T, answered in second level spectrum
Spy should be included in second level spectrum for having modified the modification peptide fragment of two molecule girard reagent T comprising characteristic peak M/z=315.16
Levy peak M/z=214.625.
The present invention has the advantage that
1. the present invention connects derivative quaternary ammonium salt group in nitrogen acetylglucosamine modified glucoprotein matter and glycopeptide in oxygen, enhance sample
Quality spectrum signal.
2. oxygen derived from the present invention connects nitrogen acetylglucosamine modified glucoprotein matter and glycopeptide, part original nitrogen acetyl is remained
The group of aminoglucose provides information for subsequent mass spectrum solution spectrum analysis.
3. the present invention is using the connection nitrogen acetylglucosamine modified glucoprotein matter of oxygen derived from chemical method and glycopeptide, more honest and cleaner
Valence is conducive to a large amount of Mass-analysis of samples.
4. the peptide fragment after the present invention is derivative can obtain preferable second level spectrogram under CID mode, it is conducive to subsequent data
Library searching analysis.
Detailed description of the invention
Fig. 1 oxygen connects nitrogen acetylglucosamine modified glucoprotein matter chemical derivatization and mass spectral analysis flow chart；
Fig. 2 oxygen connects MALDI mass spectrum after the amine-modified mark peptide TAPTSgTIAPG of nitrogen Acetylglucos derives；
The derivative front and back mass signal enhancing amplitude of Fig. 3 mark peptide TAPTSgTIAPG compare figure a) MALDI derived peptide segment with not
Derived peptide segment 1:5 mixing, b) LTQ derived peptide segment mixes with non-derived peptide segment 1:1；
Second order ms figure a) under the CID mode of peptide TAPTSgTIAPG is marked after Fig. 4 is derivative has modified the examination of two molecule Geralds
The peptide fragment second order ms figure of agent T, b) modify the peptide fragment second order ms figure of a molecule girard reagent T；
Fragmentation mode schematic diagram a) under the CID mode of peptide TAPTSgTIAPG is marked after Fig. 5 is derivative has modified the drawing of two molecule Jis
The peptide fragment second level fragmentation mode of moral reagent T, b) modify the peptide fragment second level fragmentation mode of a molecule girard reagent T.
Specific embodiment
Method provided by the invention is described in detail below by embodiment, but the invention is not limited in any way.
Embodiment 1
Oxygen connects the girard reagent label of the amine-modified standard sugar peptide of nitrogen Acetylglucos:
1. as shown in Figure 1, pressing following flow operations:
1) standard glycopeptide N-terminal di-methylation is closed: take 10 μ g peptide fragment TAPTSgTIAPG that 4% formalin, 1 μ l is added,
1 μ l of 0.6mM sodium cyanoborohydride reacts 0.5 hour, and reverse phase desalination removes formaldehyde and sodium cyanoborohydride.
2) nitrogen acetylglucosamine radical oxidation: in the 50mM sodium acetate buffer solution of pH=5.5, glycopeptide sample and concentration
For the reaction of 20mM sodium periodate solution；37 DEG C of reaction temperature, the reaction time 6 hours.After oxidation reaction, 5 times of height are added
Sodium iodate mole sodium sulfite reacts 20 minutes, terminates oxidation reaction.
3) the glycopeptide sample of sodium periodate oxidation girard reagent derivatization reaction: will be passed through in the Gerald that concentration is 50mM
Reagent T mixing, reacts 2 hours.Reaction process carries out in the 50mM sodium acetate solution of pH=5.5.Using reversed-phase liquid chromatography
It is separated off unreacted girard reagent.
2. the MALDI mass spectrogram of the mark peptide after deriving is as shown in Figure 2
3. learning that a molecule mark peptide is reacted with a molecule girard reagent T, without apparent unreacted by molecular weight calculating
Mark peptide peak.It can thus be assumed that the labeling effciency of the mark peptide is close to completely.
Embodiment 2
The enzymatic hydrolysis and label of protein example:
(1) extract after protein example carries out denaturation reductive alkylation and digest to mouse brain sample: every 100 μ g extracts egg
4 μ l 1M dithiothreitol (DTT)s (DTT) are added in white matter, and 56 DEG C are reacted 1.5 hours.8 μ l 1M iodoacetic acid (IAA) are added later, are protected from light
Reaction 30 minutes.By treated, protein example utilizes ammonium bicarbonate buffers to dilute, pH=8.Every 100 μ g protein is added
4 μ g trypsase are digested, and 37 DEG C are reacted 24 hours.Peptide hydrolysis removes small point of ammonium hydrogen carbonate etc. using reversed-phase liquid chromatography
Alite, the peptide fragment sample obtained after freeze-drying carry out formaldehyde di-methylation method closing N-terminal amino.Every 100 μ g peptide fragment adds
Enter 4% formalin, 15 μ l, 0.6mM sodium cyanoborohydride, 15 μ l, react 0.5 hour, reverse phase desalination removes formaldehyde and cyano boron
Sodium hydride.
(2) nitrogen acetylglucosamine radical oxidation: in the 50mM sodium acetate buffer solution of pH=5.5, oxygen connects nitrogen acetyl
Aminoglucose modified glucoprotein matter or glycopeptide sample are reacted with concentration for 20mM sodium periodate solution；37 DEG C of reaction temperature, when reaction
Between 6 hours.After oxidation reaction, 5 times of sodium metaperiodate mole sodium sulfites are added, react 30 minutes, it is anti-to terminate oxidation
It answers.
(3) girard reagent derivatization reaction: by by the glycoprotein sample of sodium periodate oxidation in concentration be 50mM
Girard reagent mixing, reacts 6 hours.Reaction process carries out in the sodium acetate solution of pH=5.5.
(4) unreacted girard reagent is separated off using reversed-phase liquid chromatography: is lived using reverse phase C18 or C8 chromatography, flowed
Dynamic phase A:2% acetonitrile, 98% water and 0.1% trifluoroacetic acid.Mobile phase B: 98% acetonitrile, 2% water and 0.1% trifluoroacetic acid.It adopts
With 2% Mobile phase B loading, chromatographic column 10 minutes are rinsed with sample-loading buffer later, later with 80% mobile phase by sugared egg
White matter or glycopeptide sample elution get off, freeze-drying.
Embodiment 3
To investigate influence of the quaternary ammonium salt modification group to mass signal, MALDI has been investigated with above-mentioned girard reagent T mark peptide
And the signal response condition of two kinds of ion source massspectrums of ESI:
1.MALDI mass spectrum response condition: by after chemical derivatization mark peptide fragment and the closed mark peptide of di-methylation mixed with 1:5
It closes, using MALDI mass spectral analysis, spectrogram such as Fig. 3 (a) enhances about 10 times after mass signal is derivative in conjunction with molar ratio,
2.ESI mass spectrum response condition: by after chemical derivatization mark peptide fragment and the closed mark peptide of di-methylation mixed with 1:1,
It is analyzed using RP-ESI-LTQ, spectrogram such as Fig. 3 (b), enhances about 2.2 times after mass signal is derivative.
Embodiment 4
Second level fragmentation feelings under the CID mode of nitrogen acetylglucosamine modification peptide fragment are connected to investigate the oxygen after quaternary ammonium salt derives
Condition under conditions of CID collision energy 35%, acquires second level fragmentation of ions mass spectrogram using Thermo LTQ XL mass spectrum:
The second order ms of the derivative upper two molecules girard reagent T of mark chemistry of peptides in above-described embodiment 1: the mark peptide after derivative
Three valence charge of band uses shown in spectrogram such as Fig. 4 (a) of CID mode fragmentation, in addition to the fragment ion that peptide fragment generates in spectrogram,
There are also characteristic peak ion g1, g2, g3, and the sugar-modified group that falls off peptide fragment quasi-molecular ions presence, the fragmentation mould of the modification group
Shown in formula such as Fig. 5 (a).Mark the second order ms of the derivative upper molecule girard reagent T of chemistry of peptides: since steric hindrance and electrostatic are arranged
Reprimand effect, part peptide fragment can only derive upper molecule girard reagent T, and the mark peptide band divalent charge after deriving uses CID mould
Shown in the spectrogram of formula fragmentation such as Fig. 4 (b), in addition to the fragment ion that peptide fragment generates in spectrogram, there are also characteristic peak ion g1With fall off
The presence of the peptide fragment quasi-molecular ions of sugar-modified group, shown in the fragmentation mode of the modification group such as Fig. 5 (b).
Embodiment 5
It investigates amine-modified using oxygen connection nitrogen Acetylglucos of the protein database search engine retrieval quaternary ammonium salt after derivative
The case where peptide fragment:
It is marked as embodiment 1 will mark peptide TAPTSgTIAPG using girard reagent T, later using AB5600's
RPLC-MS/MS system analyzes the peptide fragment, and the data of acquisition use mascot search engine.In data lab setting with
E.coli database is background, and artificially editor adds a new protein sequence, albumen in the fasta file of the database
Name name ecoli_add_opeptide, amino acid sequence are as follows: MRIHSPYPASWALAQRIGYLYSEGEIIYLADTPFERLL
DIQRQVGQCQTMTSLSQADFEARLEAVFHQNTGESQQIAQDIDQSVDLLSLSEEMPANEDLLNEDSAAPVIRLINA
ILSEAIKETASDIHIETYEKTMSIRFRIDGVLRTILQPNKKLAALLISRIKVMARLDIAEKRIPQDGRISLRIGRR
NIDVRVSTLPSIYGERAVLRLLDKNSLQLSLNNLGMTAADKQDLENLIQLPHGIILVTGPTGSGKSTTLYAILSAL
NTPGRNILTVEDPVEYELEGIGQTQVNTRVDMSFARGLRAILRQDPDVVMVGEIRDTETAQIAVQASLTGHLVLST
LHTNSASGAVTRLRDMGVESFLLSSSLAGIIAQRLVRRLCPQCRQFTPVSPQQAQMFKYHQLAVTTIGTPVGCPHC
HQSGYQGRMAIHEMMVVTPELRAAIHENVDEQALERLVRQQHKALIKTAPTSTIAP G, C-terminal sequence and mark peptide
TAPTSgTIAPG is identical, can be used as the database for retrieving the mark peptide.
1. the mark peptide in above-described embodiment 1 may be searched by a molecule girard reagent T, concrete database in chemical derivatization
Rope parameter is
Enzyme:Trypsin
Fixed modifications:Carbamidomethyl (C)
Variable modification:Dimethyl (K), Dimethyl (N-term),
Opeptide314 (ST), Oxidation (M)
Mass values:Monoisotopic
Protein Mass:Unrestricted
Peptide Mass Tolerance: ± 0.05Da (#13C=2)
Fragment Mass Tolerance: ± 0.1Da
Max Missed Cleavages:2
Instrument type:ESI-QUAD-TOF
Wherein the part modification except opeptide314 (ST) in addition to is built in system, selection when setting, and
The opeptide314's (ST) added afterwards is specifically configured to decorating molecule formula C13H22N4O5, decorating site serine and Soviet Union's ammonia
Sour residue.Search result are as follows: retrieve mark peptide TAPTSgTIAPG, score:26, Expect:1.5.
2. the mark peptide in above-described embodiment 1 may be searched by two molecule girard reagent T, concrete database in chemical derivatization
Rope parameter is
Enzyme:Trypsin
Fixed modifications:Carbamidomethyl (C)
Variable modification:Dimethyl (K), Dimethyl (N-term),
Opeptide427 (ST), Oxidation (M)
Mass values:Monoisotopic
Protein Mass:Unrestricted
Peptide Mass Tolerance: ± 0.05Da (#13C=2)
Fragment Mass Tolerance: ± 0.1Da
Max Missed Cleavages:2
Instrument type:ESI-QUAD-TOF
Wherein the part modification except opeptide427 (ST) in addition to is built in system, selection when setting, and
The opeptide427's (ST) added afterwards is specifically configured to decorating molecule formula C18H33N7O5, decorating site serine and Soviet Union's ammonia
Sour residue.Search result are as follows: retrieve mark peptide TAPTSgTIAPG, score:18, Expect:1.5.
Claims (4)
1. based on the analysis method of mass spectrographic oxygen connection nitrogen acetylglucosamine modified glucoprotein matter, it is characterized in that: first by O-
GlcNAc modified glucoprotein matter generates peptide fragment using protease hydrolyzed；Later, oxidant is made with sodium metaperiodate, to the nitrogen second of modification
Vicinal hydroxyl groups in acyl glucose amine groups carry out ring-opening reaction and form two aldehyde groups；Utilize the acyl trap on girard reagent T
It is reacted with newly-generated aldehyde radical, to make quaternary ammonium salt group in glycopeptide chemical derivatization；Then Mass Spectrometric Identification and analysis are carried out;
The fragment information that peptide fragment is obtained by second order ms, by analysis means such as database search to this kind of glycosyl modified sugar
Protein carries out analysis and identification;
Using LC-MS, sample analysis is carried out using mass spectrum after separating to peptide fragment；
It is broken that second level is carried out to peptide fragment using collision induced dissociation (Collision-induced dissociation, CID) mode
It splits, for ion trap mass spectrometry, CID energy hole is in 30-50%；Data are acquired to carry out using proteome databases search engine
The use of information of glycopeptide is analyzed;
Due to there is a plurality of general proteome databases search engines at present, design parameter setting is slightly different；
Therefore in the oxygen connection amine-modified parameter setting of nitrogen Acetylglucos, having of especially adjusting need to be made: 1) being increased variable
The setting of modification, decorating molecule formula C13H22N4O5, decorating site serine and threonine residues；2) increase setting for variable modification
It sets, decorating molecule formula C18H33N7O5, decorating site serine and threonine residues；3) increase variable modification: decorating molecule formula
C2H4, modify position lysine residue；4) decorating molecule formula C2H4, decorating site peptide fragment nitrogen end；Library result is searched to be further processed,
For the confidence level for increasing result, further screened using the characteristic peak in second order ms figure；For having modified molecule Ji
The modification peptide fragment of ladd reagent T, second level spectrum in should include characteristic peak M/z=315.16, for having modified two molecule girard reagents
The modification peptide fragment of T, second level spectrum in should include characteristic peak M/z=214.625.
2. analysis method described in accordance with the claim 1, it is characterized in that: nitrogen acetylglucosamine modified glucoprotein matter is being connected to oxygen
Protein digestion is carried out after denaturation reductive alkylation, obtains the peptide fragment as enzymolysis product；3-6 μ l is added in every 100 μ g protein
1 M dithiothreitol (DTT) (DTT), 50-60 DEG C reaction 1-2 hours；1 M iodoacetic acid (IAA) of 8-12 μ l is added later, is protected from light
Reaction 10-30 minutes；Treated protein example is diluted using ammonium bicarbonate buffers, pH value control 7.5-8.5 it
Between, every 100 μ g protein is added 2-5 μ g protease and is digested, 36-38 DEG C reaction 16-24 hours；Peptide hydrolysis is using instead
Phase liquid chromatogram removes the small molecule salts such as ammonium hydrogen carbonate, and the peptide fragment sample obtained after freeze-drying carries out formaldehyde di-methylation method
Close N-terminal amino.
3. analysis method according to claim 1 or 2, it is characterized in that: it is serine or threonine to avoid peptide fragment N-terminal
Existing side reaction when residue closes N-terminal amino using formaldehyde di-methylation method, and mass concentration is added in every 100 μ g peptide fragment
3-5% formalin 10-25 μ l, 0.5-0.8 mM sodium cyanoborohydride 10-25 μ l reacts 0.5-2 hours, the removal of reverse phase desalination
Formaldehyde and sodium cyanoborohydride；Glycopeptide is reacted with sodium metaperiodate later, to the ortho position hydroxyl in the nitrogen Acetylglucos amine groups of modification
Base carries out ring-opening reaction and forms two aldehyde groups；For excessive sodium metaperiodate, reducing agent is made using sodium sulfite and is terminated instead
It answers；Girard reagent T is added later to react with newly-generated aldehyde radical, excessive girard reagent T utilizes reversed-phase liquid chromatography point
From removing.
4. analysis method described in accordance with the claim 1, it is characterized in that: the amine-modified glycopeptide reaction of nitrogen Acetylglucos is connect with oxygen
Sodium periodate solution concentration is 20-30 mM；35-40 DEG C of reaction temperature, reaction time 4-8 hour；After oxidation reaction, add
Enter the sodium sulfite of 4-6 times of sodium metaperiodate mole, react 10-40 minutes, terminates oxidation reaction；Whole process is in pH=5-
It is carried out in 6 sodium acetate solution；It will be mixed by the glycopeptide of sodium periodate oxidation with concentration for the girard reagent of 10-100 mM
It closes, reacts 1-6 hours；Reaction process carries out in the sodium acetate solution of pH=5-6；It is separated off not using reversed-phase liquid chromatography
React girard reagent.
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