CN112285249A - Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry - Google Patents
Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry Download PDFInfo
- Publication number
- CN112285249A CN112285249A CN201910669704.0A CN201910669704A CN112285249A CN 112285249 A CN112285249 A CN 112285249A CN 201910669704 A CN201910669704 A CN 201910669704A CN 112285249 A CN112285249 A CN 112285249A
- Authority
- CN
- China
- Prior art keywords
- tfa
- solution
- sample
- glycopeptide
- acn
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G01N2030/067—Preparation by reaction, e.g. derivatising the sample
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
Abstract
The invention belongs to the field of glycoproteomics analysis, and relates to a method for complete glycopeptide derivatization and charge transfer fragmentation mass spectrometry; the method comprises the steps of performing tertiary amine micromolecule derivatization on all carboxyl groups of the N-glycopeptide, and then sending the N-glycopeptide into charge transfer fragmentation mass spectrometry. The method has simple steps, convenient operation, rapidness and high efficiency, and can realize high-sensitivity and high-reliability analysis of the complete glycopeptide.
Description
Technical Field
The invention belongs to the field of glycoproteomics analysis, and relates to a method for complete glycopeptide derivatization and charge transfer fragmentation mass spectrometry. The method can obviously improve the analysis efficiency of the complete glycopeptide charge transfer fragmentation mass spectrometry, and has the characteristics of simple steps, convenient and quick operation and the like.
Background
Protein N-glycosylation is a ubiquitous post-translational modification in organisms. Glycosylation modification plays an important role in cell recognition and molecular recognition, protein folding, and maintenance of the correct conformation of proteins. The complete glycopeptide analysis can not only obtain the structural information of the sugar chain, but also obtain the information such as glycosylation sites, site occupancy rate and the like; therefore, the precise analysis of complete glycopeptides is increasingly important. The structural analysis of the complete N-glycopeptide comprises analysis of a glycosylation site, a peptide fragment sequence and a sugar chain composition, and a charge transfer fragmentation (ETD) mass spectrum can retain complete sugar chain composition information and simultaneously provide peptide fragment composition information and glycosylation site information, thereby showing great advantages in the mass spectrum analysis of the complete N-glycopeptide; however, when the charge density of the parent ion is low (z < +3 or m/z >850), the daughter ions generated by ETD fragmentation are collected together, resulting in ineffective ETD fragmentation, which limits the application of ETD in the complete N-glycopeptide analysis, therefore, improving the ETD fragmentation efficiency has become a bottleneck problem to be solved in the complete N-glycopeptide ETD analysis.
The inventor of the application intends to provide a method for improving the charge density of the complete N-glycopeptide in mass spectrometry so as to improve the ETD fragmentation efficiency of the complete N-glycopeptide, and the method is favorable for realizing accurate mass spectrometry identification of the complete glycopeptide, thereby further promoting the research of glycoproteomics.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a mass spectrometric analysis method for derivation and charge transfer fragmentation of complete glycopeptides; the method can obviously improve the analysis efficiency of the charge transfer fragmentation mass spectrometry of the complete glycopeptide, and has the characteristics of simple steps, convenient operation, rapidness and the like.
The invention relates to a mass spectrometric analysis method of complete glycopeptide derivation and charge transfer fragmentation, which utilizes amidation reaction between amino of tertiary amine micromolecule N, N-dimethyl ethylenediamine (DMEN) and carboxyl of peptide segment and carboxyl of sugar chain sialic acid in the complete glycopeptide to introduce the tertiary amine micromolecule into the complete glycopeptide, thereby realizing improvement of the charge number of the complete glycopeptide in mass spectrometric analysis and improving ETD fragmentation efficiency of the glycopeptide; the method comprises the following steps:
(1) carrying out trypsin enzymolysis treatment on the protein sample;
(2) carrying out dimethylation marking on the sample after enzymolysis;
(3) derivatization and use of the complete glycopeptide with tertiary amine small molecule N, N-dimethylethylenediamine
Enriching ZIC-HILIC;
(4) and (4) sending the complete glycopeptide obtained by enrichment to mass spectrometry.
Specifically, the method of the invention utilizes the reaction of tertiary amine micromolecule N, N-dimethyl ethylenediamine and carboxyl on the complete glycopeptide to mark DMEN on the complete glycopeptide, thereby improving the charge number of the complete glycopeptide in mass spectrometry and finally improving the ETD fragmentation efficiency of the complete glycopeptide; the method is characterized in that an amidation reaction between amino of N, N-dimethyl ethylenediamine and carboxyl of acidic amino acid is adopted to introduce tertiary amine micromolecules onto the complete glycopeptide, so that the charge number of the complete glycopeptide in mass spectrometry is improved, the ETD fragmentation efficiency of the complete glycopeptide is improved, and the analysis of the complete glycopeptide in a sample is realized, and the method comprises the following steps:
(1) carrying out reductive alkylation reaction on the protein sample to open the disulfide bond of the protein;
(2) carrying out enzymolysis treatment on the sample by using trypsin, and carrying out enzymolysis on the protein into peptide fragments;
(3) carrying out dimethyl derivatization on the enzymolysis peptide segment to protect the N end of the peptide segment and the amino group of the side chain;
(4) c18SPE desalting is carried out on the reacted peptide segment, and redundant reaction reagents are removed;
(5) carrying out tertiary amine micromolecule N, N-dimethylethylenediamine derivatization on a sample;
(6) enriching complete glycopeptides by using a ZIC-HILIC material and removing a derivative reagent;
(7) and (4) sending the complete glycopeptide obtained by enrichment into LC-MS (liquid chromatography-mass spectrometry) for analysis.
More specifically, in the method, carboxyl on the complete glycopeptide is reacted with tertiary amine micromolecule N, N-dimethyl ethylenediamine, so that the charge number of the complete glycopeptide in mass spectrometry is improved, the ETD fragmentation efficiency of the complete glycopeptide is improved, and qualitative and quantitative mass spectrometry of the complete glycopeptide in a sample is realized;
in the step (1), the protein sample with the concentration of 0.5-1.0 mu g/mu L is dissolved in 100mM TEAB solution; the final concentration of DTT and IAA in the protein sample solution was 10mM and 20 mM;
dissolving 100 μ g protein with 200 μ L100 mM triethylammonium bicarbonate buffer (TEAB), heat denaturing, adding 2 μ L1M Dithiothreitol (DTT) solution, and incubating at 56 deg.C for 1 h; then adding 4 mu L of 1M Iodoacetamide (IAA) solution to react for 45min at room temperature in a dark place;
in the step (2), 2 mug of trypsin (enzyme: protein, 1: 50, mass ratio) is added into the protein sample, 1 mug of trypsin (enzyme: protein, 1:100, mass ratio) is added after 12 hours of enzymolysis at 37 ℃, and then 2 hours of enzymolysis is carried out; after the enzymolysis of the protein is finished, putting the mixture into a water bath at 100 ℃ for incubation for 5min to stop the enzymolysis;
in the step (3), 17 μ L of 4% formaldehyde solution ((HCHO, DCDO or D) is added into 100 μ g of the enzymolyzed peptide fragment solution13CDO), mixing, adding 17 μ L of 0.60M sodium cyanoborohydride (NaCNBH)3Or NaCNBD3) Reacting the solution at 37 ℃ for 2 h;
in the step (4), C18SPE desalting is performed on the sample after the dimethylation, and the concrete desalting is as follows:
activating a C18 desalting column with 1mL of ACN containing 0.10% trifluoroacetic acid (TFA);
② a desalting column of 1mL of ultrapure water containing 0.10 percent TFA balanced C18;
③ loading the sample, adding the enzymatic hydrolysis peptide segment which is marked by dimethyl into a C18SPE desalting column, and leading the enzymatic hydrolysis peptide segment to pass through a C18 stationary phase under the action of gravity; collecting the sample liquid, and repeatedly sampling once;
fourthly, washing the C18 desalting column by using 3mL of ultrapure water containing 0.10% TFA, and removing impurities such as salt;
using 300 μ L H containing 0.10% TFA2Eluting the peptide fragment with O/ACN (60/40, v/v) eluent, and collecting the solution;
sixthly, the mixture is treated by 300 mu L of H containing 0.10 percent TFA2Eluting the peptide fragment with O/ACN (40/60, v/v) eluent, and combining the two eluates;
seventhly, freeze-drying the collected eluent, and carrying out the next amidation reaction;
in the step (5), the sample needs to be freeze-dried and kept in an anhydrous state; re-dissolving the sample with DMSO; the concentration of DMEN is 5M and the concentration of PyAOP is 400 mM; preparing a reaction reagent by DMSO; the reaction time is 2h, and the reaction temperature is room temperature;
the specific steps are, firstly, re-dissolving the freeze-dried sample with 29 μ L dimethyl sulfoxide (DMSO), then adding 1 μ L1M TEAB, 5 μ L5M DMEN, 2.5 μ L4-methylmorpholine (NMM) in sequence, mixing them uniformly, then adding 12.5 μ L400 mM hexafluorophosphate (7-azabenzotriazole-1-oxy) tripyrrolidinophosphonium (PyAOP) solution (dissolved in DMSO); after fully mixing, placing the reaction solution at room temperature for suspension reaction for 2 hours; after the reaction was complete, 800. mu.L of ACN/H was added2Mixing O/TFA (80/19/1, v/v/v) solution, and enriching ZIC-HILIC;
in the step (6), 800 μ LACN/H is added to the reacted sample in the step (5)2Mixing O/TFA (80/19/1, v/v/v) solution, and enriching ZIC-HILIC;
the ZIC-HILIC enrichment specific steps are as follows:
preparing a ZIC-HILIC small column: firstly, blocking a 200L gun head by using a small cotton tuft, and then, erecting the gun head on a 2mL test tube by using a plastic ring; weighing 30mg of ZIC-HILIC chromatographic filler, dispersing the ZIC-HILIC chromatographic filler in 50% ACN solution containing 0.10% TFA, uniformly mixing, adding the mixture into a 200L gun head, and centrifugally compacting the filler;
secondly, activating a ZIC-HILIC small column by using 200L of ultrapure water containing 0.10 percent TFA, and centrifuging for 2min at 2500rcf to enable the solution to flow through the filler in the gun head; repeating twice;
③ using 200L ACN/H2Equilibrating the ZIC-HILIC column with O/TFA (80/19/1, v/v/v) solution, centrifuging at 3500rcf for 1min, and flowing the solution through the packing in the tip; repeating for three times;
fourthly, adding the peptide segment marked by the DMEN into a ZIC-HILIC column, and centrifuging for 1.5min at 3500 rcf; loading 300L of sample each time until the sample is added;
using 200L ACN/H2Washing ZIC-HILIC column with O/TFA (80/19/1, v/v/v) solution, centrifuging at 3500rcf for 1min, and removing non-specific adsorption such as peptide fragment and salt; repeating for three times;
sixthly, the complete N-glycopeptide is eluted by 100L of ultrapure water containing 0.10 percent of TFA; repeating for three times, and combining eluates;
seventhly, freeze-drying the collected eluent, and performing mass spectrometry after redissolving;
in the step (7), the sample obtained by enrichment in the step (6) is sent to LC-MS analysis and detection.
In the method, the tertiary amine N, N-dimethylethylenediamine is marked on the complete N-glycopeptide, so that the charge number of the complete N-glycopeptide in mass spectrometry can be obviously improved, the ETD fragmentation efficiency of the complete N-glycopeptide is improved, and the aim of quantitatively analyzing the complete glycopeptide can be simultaneously fulfilled by using the dimethylated light and heavy isotope labels.
Drawings
FIG. 1 is a flow chart of the method.
FIG. 2 is a MALDI-TOF mass spectrum of intact N-glycopeptide of immunoglobulin (IgG) before and after derivatization, with the ordinate of the MALDI-TOF-MS spectrum being the relative Intensity of the mass peak (% Intensity) and the abscissa being the mass-to-charge ratio (m/z): (a) before the derivation, (b) is derived from DMEN. Paired intact N-glycopeptides from IgG2 and IgG1, G0F had a sugar chain composition of HexNAc4Hex3Fuc1, G1F had a sugar chain composition of HexNAc4Hex4Fuc1, G2F had a sugar chain composition of HexNAc4Hex5Fuc1, G1FS had a sugar chain composition of HexNAc4Hex4Fuc1NeuAc1, and G2FS had a sugar chain composition of HexNAc4Hex5Fuc1NeuAc 1. Comparing panel (a) to panel (b) it can be seen that unreacted IgG intact N-glycopeptide was not detected in the derived spectra, indicating that the efficiency of the derivatization of intact N-glycopeptide is close to 100%.
FIG. 3 is a LC-ESI mass spectrum of IgG intact N-glycopeptide, plotted on the ordinate versus the relative Intensity of the mass peak (% Intensity) and on the abscissa, the mass to charge ratio (m/z): (a, b) underivatized, (c, d) dimethylated, (e, f) DMEN-tagged. As can be seen from the comparison, when the complete N-glycopeptide is labeled by DMEN, the number of charges carried by the complete N-glycopeptide is obviously increased, and the complete N-glycopeptide mainly has charges of +3 and +4 and has a lower proportion of charges of + 5. For the acid complete N-glycopeptide containing sialic acid, due to the fact that DMEN derivatization occurs on carboxyl, more DMEN molecules are marked on the acid complete N-glycopeptide, the charge number is improved more obviously, and the charge number is improved to +3, +4, +5 and + 6.
FIG. 4 is an ETD-MSMS tandem mass spectrum of DMEN labeled IgG whole N-glycopeptide with the ordinate of the spectrum being the relative Intensity of the mass peak (% Intensity) and the abscissa of the spectrum being the mass to charge ratio (m/z): (a) IgG2-G0F, [ M +4H ]4+, M/z 710.85, (b) IgG2-G1, [ M +4H ]4+, M/z 714.84, (c) IgG3/4-G0F, [ M +4H ]4+, M/z 714.84 and (d) IgG1-G2FS, [ M +5H ]5+, M/z 712.33. ETD fragmentation can achieve 100% peptide sequence coverage, thus enabling clear identification and differentiation of these intact N-glycopeptides. Meanwhile, four subtypes of IgG can be distinguished through c \ z ions. Therefore, the DMEN mark can obviously improve the ETD fragmentation efficiency of IgG complete N-glycopeptide, improve the accuracy and reliability of complete N-glycopeptide identification, and is favorable for distinguishing isomers.
FIG. 5 is an ETD-MSMS mass spectrum of the entire N-glycopeptide of the DMEN-labeled glycoprotein bovine Fetuin (Fetuin) with the ordinate of the spectrum showing the relative Intensity of the mass peaks (% Intensity) and the abscissa of the spectrum showing the mass to charge ratio (m/z): (a) [ M +7H ]7+, M/z 722.35, (b) [ M +8H ]8+, M/z 800.41, (c) [ M +10H ]10+, M/z 727.08 and (d) [ M +9H ]9+, M/z 727.05. For the fetuin complete N-glycopeptide with a longer peptide segment length, the charge number of the parent ion of the fetuin complete N-glycopeptide can be obviously improved after the DMEN is marked.
Detailed Description
The following example is a further illustration of the method of derivatization and charge transfer fragmentation mass spectrometry of an intact glycopeptide set forth herein.
EXAMPLE 1 DMEN derivatization of intact glycopeptides
Mu.g of IgG protein was dissolved in DMSO, then 1. mu.L of 1M TEAB, 5. mu.L of 5M DMEN, 2.5. mu.L of NMM were added in sequence, mixed well, followed by 12.5. mu.L of 400mM PyAOP solution (in DMSO). After thorough mixing, the reaction solution was left to stand at room temperature for suspension reaction for 2 hours. After the reaction was complete, 800. mu.L of ACN/H was added2And mixing O/TFA (80/19/1, v/v/v) solution, and performing ZIC-HILIC enrichment. MALDI-TOF-MS analysis of the enriched solution (final IgG intact glycopeptide-containing solution) spot target is shown in FIG. 2.
Example 2 experiment of charge number enhancement in electrospray mass spectrometry of DMEN-derived intact glycopeptides
100. mu.g of IgG protein was dissolved in DMSO. Then 1. mu.L of 1M TEAB, 5. mu.L of 5M DMEN, 2.5. mu.L of NMM were added in succession, mixed well and then 12.5. mu.L of 400mM PyAOP solution (in DMSO) was added. After mixing thoroughly, the reaction solution is placedThe reaction was suspended at room temperature for 2 h. After the reaction was complete, 800. mu.L of ACN/H was added2Mixing O/TFA (80/19/1, v/v/v) solution, enriching ZIC-HILIC, freeze-drying the enriched solution, and analyzing and detecting by LC-ESI-MS in ETD mode; the results are shown in FIG. 3.
Example 3 experiments on enhanced mass spectrometric charge transfer fragmentation efficiency of DMEN-derived intact glycopeptides
1. 100 μ g of standard IgG protein (IgG) was dissolved in DMSO. Then 1. mu.L of 1M TEAB, 5. mu.L of 5M DMEN, 2.5. mu.L of NMM were added in succession, mixed well and then 12.5. mu.L of 400mM PyAOP solution (in DMSO) was added. After thorough mixing, the reaction solution was left to stand at room temperature for suspension reaction for 2 hours. After the reaction is finished, adding 800 mu L of ACN/H2O/TFA (80/19/1, v/v/v) solution, uniformly mixing, carrying out ZIC-HILIC enrichment, freeze-drying the solution, and then carrying out analysis and detection by using LC-ESI-MS by adopting an ETD-MSMS mode; the results are shown in FIG. 4.
2. Dissolve 100. mu.g of Fetuin in DMSO. Then 1. mu.L of 1M TEAB, 5. mu.L of 5M DMEN, 2.5. mu.L of NMM were added in succession, mixed well and then 12.5. mu.L of 400mM PyAOP solution (in DMSO) was added. After thorough mixing, the reaction solution was left to stand at room temperature for suspension reaction for 2 hours. After the reaction is finished, adding 800 mu L of ACN/H2O/TFA (80/19/1, v/v/v) solution, uniformly mixing, carrying out ZIC-HILIC enrichment, freeze-drying the solution, and then carrying out analysis and detection by using LC-ESI-MS by adopting an ETD-MSMS mode; the results are shown in FIG. 5.
Claims (18)
1. A method for deriving complete glycopeptide and analyzing charge transfer fragmentation mass spectrometry is characterized in that the tertiary amine small molecule DMEN is introduced to the complete glycopeptide by amidation reaction between amino of N, N-dimethyl ethylenediamine (DMEN) and carboxyl of acidic amino acid, and then analysis is carried out; the method comprises the following steps:
(1) carrying out reductive alkylation reaction on the protein sample to open the disulfide bond of the protein;
(2) carrying out enzymolysis treatment on the sample by using trypsin, and carrying out enzymolysis on the protein into peptide fragments;
(3) carrying out dimethyl derivatization on the enzymolysis peptide segment to protect the amino at the N end of the peptide segment and the amino of a side chain;
(4) c18SPE desalting is carried out on the reacted peptide segment, and redundant reaction reagents are removed;
(5) carrying out tertiary amine micromolecule N, N-dimethylethylenediamine derivatization on a sample;
(6) enriching intact glycopeptides with a ZIC-HILIC material and removing excess derivatizing reagent;
(7) and (4) sending the complete glycopeptide obtained by enrichment into LC-MS, and analyzing by adopting an ETD mode.
2. The method of claim 1, wherein in step (1), the protein sample is dissolved in 100mM TEAB solution at a concentration of 0.5-1.0 μ g/μ L.
3. The method of claim 1, wherein in step (1), the final concentration of DTT and the final concentration of IAA in the protein sample solution are 10mM and 20mM, respectively.
4. The method of claim 1, wherein in step (1), the reaction time of DTT is 1h, and the reaction temperature is 56 ℃; the reaction time of IAA is 45min, the reaction is protected from light, and the reaction temperature is room temperature.
5. The method of claim 1, wherein in step (2), the mass ratio of the protein sample to trypsin is 1: 50 and 1: 100.
6. The method of claim 1, wherein in step (3), a formaldehyde solution (HCHO, DCDO, or D) is added13CDO) 4% by volume, sodium cyanoborohydride solution (NaCNBH)3Or NaCNBD3) The concentration of (3) is 0.60M.
7. The process according to claim 1, wherein in step (3), the reaction time is 2 hours and the reaction temperature is 37 ℃.
8. The method of claim 1, wherein, in step (4),the temperature of desalting by C18-SPE is 25-37 ℃; the activating solution was ACN with 0.10% TFA and the equilibrium solution was H with 0.10% TFA2O; cleaning solution H of 0.10% TFA2O; elution solution H of 0.10% TFA2O/ACN (60/40, v/v), and H with 0.10% TFA2O/ACN(40/60，v/v)。
9. The method of claim 8 wherein in step (4) the dimethylated sample is subjected to C18SPE desalination by the steps of:
activating a C18 desalting column with 1mL of ACN containing 0.10% trifluoroacetic acid (TFA);
② a desalting column of 1mL of ultrapure water containing 0.10 percent TFA balanced C18;
③ loading the sample, adding the enzymatic hydrolysis peptide segment which is marked by dimethyl into a C18SPE desalting column, and leading the enzymatic hydrolysis peptide segment to pass through a C18 stationary phase under the action of gravity; collecting the sample liquid, and repeatedly sampling once;
fourthly, washing the C18 desalting column by using 3mL of ultrapure water containing 0.10% TFA, and removing impurities such as salt;
using 300L H containing 0.10% TFA2Eluting the peptide fragment with O/ACN (60/40, v/v) eluent, and collecting the solution;
sixthly, using 300L H containing 0.10% TFA2Eluting the peptide fragment with O/ACN (40/60, v/v) eluent, and combining the two eluates;
and seventhly, freeze-drying the collected eluent, and carrying out the next amidation reaction.
10. The method of claim 1, wherein in step (5), the sample is lyophilized and maintained in an anhydrous state.
11. The method of claim 1, wherein in step (5), the sample is reconstituted with DMSO.
12. The method according to claim 1, wherein in step (5) the concentration of DMEN is 5M and the concentration of PyAOP is 400 mM.
13. The method of claim 1, wherein in step (5), the reagent is dispensed in DMSO.
14. The method of claim 1, wherein in step (5), the reaction time is 2 hours and the reaction temperature is room temperature.
15. The method of claim 1, wherein in step (6), ACN/H is used2O/TFA (80/19/1, v/v/v) solution was used to dilute the reacted sample 20-fold.
16. The method of claim 1, wherein in step (6), the column is prepared using 30mg of ZIC-HILIC chromatography packing.
17. The method of claim 1, wherein in step (6), the column is prepared using 30mg of ZIC-HILIC chromatography packing; the activating solution was H with 0.10% TFA2O, the equilibrium solution is ACN/H2O/TFA (80/19/1, v/v/v); the cleaning solution is ACN/H2O/TFA (80/19/1, v/v/v); the elution solution was an aqueous solution containing 0.10% TFA.
18. The method of claim 1, wherein in step (7), the LC-MS scanning mode is ETD mode.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910669704.0A CN112285249A (en) | 2019-07-24 | 2019-07-24 | Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910669704.0A CN112285249A (en) | 2019-07-24 | 2019-07-24 | Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112285249A true CN112285249A (en) | 2021-01-29 |
Family
ID=74419399
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910669704.0A Pending CN112285249A (en) | 2019-07-24 | 2019-07-24 | Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112285249A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961060A (en) * | 2021-02-09 | 2021-06-15 | 武汉大学 | Isotope labeled N, N-dimethylethylenediamine, preparation method thereof and analysis method of short-chain fatty acid |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5432093A (en) * | 1992-11-23 | 1995-07-11 | City Of Hope | Sequential degradation of proteins and peptides from the N-terminus |
| JP2013068594A (en) * | 2011-09-05 | 2013-04-18 | Sumitomo Bakelite Co Ltd | Amidation modification method of sialo-sugar chain |
| JP2014156409A (en) * | 2013-02-14 | 2014-08-28 | Kanazawa Univ | Production method of n-type glycoprotein |
| US20170276686A1 (en) * | 2014-09-15 | 2017-09-28 | Board Of Regents, The University Of Texas System | Single molecule peptide sequencing |
| CN108548876A (en) * | 2018-03-30 | 2018-09-18 | 武汉光谷创赢生物技术开发有限公司 | The identification of phosphated peptide section and quantitative approach in a kind of improved biological sample |
-
2019
- 2019-07-24 CN CN201910669704.0A patent/CN112285249A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5432093A (en) * | 1992-11-23 | 1995-07-11 | City Of Hope | Sequential degradation of proteins and peptides from the N-terminus |
| JP2013068594A (en) * | 2011-09-05 | 2013-04-18 | Sumitomo Bakelite Co Ltd | Amidation modification method of sialo-sugar chain |
| JP2014156409A (en) * | 2013-02-14 | 2014-08-28 | Kanazawa Univ | Production method of n-type glycoprotein |
| US20170276686A1 (en) * | 2014-09-15 | 2017-09-28 | Board Of Regents, The University Of Texas System | Single molecule peptide sequencing |
| CN108548876A (en) * | 2018-03-30 | 2018-09-18 | 武汉光谷创赢生物技术开发有限公司 | The identification of phosphated peptide section and quantitative approach in a kind of improved biological sample |
Non-Patent Citations (6)
| Title |
|---|
| BRIAN L. FREY 等: "Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation", AMERICAN SOCIETY FOR MASS SPECTROMETRY * |
| BRIAN L. FREY 等: "Chemical Derivatization of Peptide Carboxyl Groups for Highly Efficient Electron Transfer Dissociation", JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY * |
| TAKASHI NISHIKAZE 等: "In-Depth Structural Characterization of N-Linked Glycopeptides Using Complete Derivatization for Carboxyl Groups Followed by Positive- and Negative-Ion Tandem Mass Spectrometry", ANALYTICAL CHEMISTRY * |
| TAKASHI NISHIKAZE 等: "In-Depth Structural Characterization of N‑Linked Glycopeptides Using Complete Derivatization for Carboxyl Groups Followed by Positive- and Negative-Ion Tandem Mass Spectrometry", ANALYTICAL CHEMISTRY * |
| 姜中尧: "利用定量化学蛋白组学方法探究硒啉类药物的靶向蛋白", 中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑 * |
| 江静 等: "亲水相互作用色谱结合串联质谱多重碎裂模式在完整糖肽研究中的应用", 分析化学 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112961060A (en) * | 2021-02-09 | 2021-06-15 | 武汉大学 | Isotope labeled N, N-dimethylethylenediamine, preparation method thereof and analysis method of short-chain fatty acid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Saba et al. | Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation | |
| Nishikaze | Sialic acid derivatization for glycan analysis by mass spectrometry | |
| CA2461587C (en) | Materials and methods for controlling isotope effects during fractionation of analytes | |
| CN105301119B (en) | Protein amino acid sequence de novo sequencing method based on the heavy label such as two ends are non- | |
| CN111381043B (en) | Sample pretreatment composition suitable for mass spectrometry detection, sample pretreatment method and application | |
| Zhang et al. | Mass spectrometry for protein sialoglycosylation | |
| AU2002341862A1 (en) | Controlling isotope effects during fractionation of analytes | |
| Aebersold et al. | Design, synthesis, and characterization of a protein sequencing reagent yielding amino acid derivatives with enhanced detectability by mass spectrometry | |
| CN109900815B (en) | Absolute quantitative analysis of IgG glycopeptides in serum | |
| EP1617223A2 (en) | Serial derivatization of peptides for "de Novo" sequencing using tandem mass spectrometry | |
| CN104237363A (en) | Protein quantification method | |
| CN106855543A (en) | A kind of protein isotopic dilution tandem mass spectrum detection method based on chemical labeling techniques | |
| Jiao et al. | Hydrazinonicotinic acid as a novel matrix for highly sensitive and selective MALDI-MS analysis of oligosaccharides | |
| CN112285249A (en) | Method for complete glycopeptide derivation and charge transfer fragmentation mass spectrometry | |
| CN111381044B (en) | Pretreatment composition, pretreatment method and application of sugar chain structure abnormal protein suitable for mass spectrometry detection | |
| CN106645437A (en) | Polypeptide amino acid sequence De novo sequencing method based on chemical modification and isotope labeling | |
| Shively et al. | Highlights of protein structural analysis | |
| CN104597187A (en) | Method for rapidly and comprehensively detecting oligosaccharide on medicinal monoclonal antibody N-glycosylation site | |
| Wang et al. | Glycan reductive amino acid coded affinity tagging (GRACAT) for highly specific analysis of N-glycome by mass spectrometry | |
| CN105445358B (en) | Analysis method based on mass spectrographic oxygen connection nitrogen acetylglucosamine modified glucoprotein matter | |
| Kaufmann et al. | Matrix-assisted laser desorption ionization (MALDI) and post-source decay (PSD) product ion mass analysis localize a photolabel crosslinked to the delta-subunit of nAChR protein by neurotoxin II | |
| Jie et al. | Highly efficient enrichment method for human plasma glycoproteome analyses using tandem hydrophilic interaction liquid chromatography workflow | |
| Francesconi | Applications of liquid chromatography–electrospray ionization‐single quadrupole mass spectrometry for determining arsenic compounds in biological samples | |
| US20050064515A1 (en) | Reagent kit of global analysis for protein expression and method for qualitative and quantitative proteomic analysis using the same | |
| Liu et al. | Discovery of Elaphuri Davidiani Cornu–specific peptide biomarkers by peptidomics analysis–based method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |