A method for analyzing protein O-glycosylation sites

A technology for glycosylation sites and proteins, applied in the field of analysis of protein O-glycosylation sites, can solve problems such as no discovery, side reactions, complex O-sugar chain structure, etc., and achieve the effect of simple method

CN104198613BActive Publication Date: 2016-04-06SHANDONG UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2016-04-06

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Abstract

The invention relates to a method for analyzing protein O-glycosylation sites. The method comprises the following steps: (1), sequentially carrying out endonuclease digestion, endoglycosidase and exoglycosidase digestion on a proteome sample or a single protein sample to prepare a peptide and non-glycopeptide protein sample or single protein sample with a GalNAc label; (2) loading the sample on a durian agglutinin chromatographic column and separating to prepare a proteomic sample; (3) separating the single protein sample or the proteomic sample through with a C18 reversed-phase chromatographic column, and then detecting by a high-definition mass spectrometer in a cation mode to obtain a high-definition mass spectrogram; (4) processing the obtained mass spectrometric data and obtaining protein glycosylation site information though the searched result. The site information can be more rapidly, more accurately and more comprehensively obtained through establishing the method for detecting the O-glycosylation sites of glycoproteins in the single protein sample and the proteomic sample.
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Description

technical field

[0001] The invention relates to a method for analyzing protein O-glycosylation sites, in particular to a method for partially degrading sugar chains by endonuclease and exonuclease combined with liquid chromatography-mass spectrometry to analyze glycoprotein O-glycosylation sites The method for detecting and identifying belongs to the technical field of biotechnology. Background technique

[0002] Protein glycosylation is an important post-translational modification. There are mainly two forms of N-glycosylation (N-glycosylation) and O-glycosylation (O-glycosylation). Protein glycosylation is involved in many cellular processes and plays an important role. In addition, the occurrence and development of some diseases such as cancer are often accompanied by abnormal changes in protein glycosylation sites and sugar chains. Therefore, the analysis of glycosylation sites is crucial for revealing abnormal changes in glycoproteins. Site analysis can not only dir...

Examples

Embodiment Construction

[0007] A method for analyzing protein O-glycosylation sites, the steps are as follows:

[0008] (1) After the proteome sample or a single protein sample is cleaved with endonuclease, the N-glycosidase and exoglycosidase are combined to cut off the N-sugar chain and part of the O-sugar chain, and dried under reduced pressure to obtain the Omics protein samples or single protein samples of GalNAc sugar-tagged peptides and non-glycopeptides;

[0009] (2) Load the omics protein samples with GalNAc sugar tags and non-glycopeptides prepared in step (1) on the durian lectin column for separation, and collect the enzyme-digested peptides with GalNAc sugar tags The peptide solution is dried under reduced pressure to prepare the hismic protein sample;

[0010] (3) Use the single protein sample prepared in step (1) or the hismic protein sample prepared in step (2) to 18 Separation by reversed-phase chromatographic column, and then detection by high-resolution mass spectrometer in posit...