Identification method for O-glycosylation peptide fragment and complete saccharide chain thereof

An identification method and glycosylation technology, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of fragmentation of the peptide sequence skeleton, poor spectrum quality, neutral loss, etc., and achieve simple and fast operation and high sensitivity High, the effect of improving sensitivity

Active Publication Date: 2016-04-06
ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI +1
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Problems solved by technology

The two fragmentation methods of LTQ-CID and HCD mainly fragment the glycosidic bond between the monosaccharides in the sugar chain, but cannot fragment the backbone of the peptide sequence, so only the information of the sugar chain composition can be obtained
Although ETD can obtain some peptide sequence information, the quality of the spectrum is relatively poor. When sialic acid exists in the O-glycopeptide, a strong neutral loss will occur, so that the scoring value of the peptide identification during the database search is very low. Low

Method used

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  • Identification method for O-glycosylation peptide fragment and complete saccharide chain thereof

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Embodiment 1

[0011] O-glycosylation analysis of embodiment 1 bovine fetuin:

[0012] 1. Add 100 μg bovine fetuin standard sample to a 0.5mL ultrafiltration tube with a molecular weight cut-off of 10kDa, add 300μL 8M urea / 100mM NH 4 HCO 3 , ultrafiltered at a centrifugal force of 14000g for 15 minutes, and with 300μL 8M urea / 100mMNH 4 HCO 3 Repeat ultrafiltration once.

[0013] 2. Add 10 μL of 1MDTT to the above ultrafiltration tube, react at 37°C for 2 hours, then add 3.8 mg of IAA, and react for 40 minutes at room temperature in the dark. Remove excess DTT or IAA by ultrafiltration for 15 min, and wash with 400 μL HO 2 O was washed twice, ultrafiltration was performed for 15 minutes each time, and the centrifugal force was 14000g.

[0014] 3. After the ultrafiltration is completed, add PNGaseF glycosidase 2 μL to the protein sample in the above ultrafiltration tube, and react overnight at 37°C. After the reaction was completed, ultrafilter again, and add 400 μL HO 2 O was washed tw...

Embodiment 2

[0022] O-glycosylation analysis of embodiment 2 human serum albumin

[0023] 1. Add 60 μL of human serum sample to a 0.5 mL ultrafiltration tube with a molecular weight cut-off of 10 kDa, add 300 μL of 8M urea / 100mM NH 4 HCO 3 , ultrafiltered at a centrifugal force of 14000g for 15 minutes, and with 300μL 8M urea / 100mMNH 4 HCO 3 Repeat ultrafiltration once;

[0024] 2. Add 10 μL of 1MDTT to the above ultrafiltration tube, react at 37°C for 2 hours, then add 3.8 mg of IAA, and react for 40 minutes at room temperature in the dark. Remove excess DTT or IAA by ultrafiltration for 15 min, and wash with 400 μL HO 2 O was washed twice, ultrafiltration was performed for 15 minutes each time, and the centrifugal force was 14000g.

[0025] 3. After the ultrafiltration is completed, add PNGaseF glycosidase 2 μL to the protein sample in the above ultrafiltration tube, and react overnight at 37°C. After the reaction was completed, ultrafilter again, and add 400 μL HO 2 O was washed ...

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Abstract

The present invention relates to an identification method for an O-glycosylation peptide fragment and a complete saccharide chain thereof. The technical process specifically comprises that a glycosylation protein sample is subjected to PNGase F digestion to remove N-glycosylation modification interference, enrichment is performed through hydrophilic interaction chromatography, and finally analysis is performed through time of flight mass spectrometer. According to the present invention, the method process has advantages of simple and rapid operation, high identification sensitivity, and the like; and the O-glycosylation peptide fragment and the complete saccharide chain thereof of the complex biological sample can be identified so as to achieve the research on the micro-uniformity of the O-glycosylation modification structure.

Description

technical field [0001] The invention relates to a method for identifying O-glycosylated peptides and their complete sugar chains. Specifically, PNGaseF enzyme treatment is performed on samples to remove interference from N-glycosylated modifications, and then enzymolysis is performed. The enzymatically hydrolyzed peptides were enriched by hydrophilic interaction chromatography and then entered into mass spectrometry analysis. The collision-induced dissociation mode of Triple-TOF mass spectrometer was used to obtain rich fragment information for database search, and finally the complete O-glycosylation modified peptide was obtained. Identification results. Background technique [0002] O-glycosylation is a common protein glycosylation modification. Studies have shown that O-glycosylation plays an important role in cell adhesion, cell signal communication, immune response and protein conversion enzyme processing, and is also associated with cancer growth and Transfer is close...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/88G01N30/08
Inventor 邹汉法朱俊程凯叶明亮
Owner ZHANGJIAGANG IND TECH RES INST CO LTD DALIAN INST OF CHEM PHYSICS CHINESE ACADEMY OF SCI
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