A method for analyzing protein O-glycosylation sites

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.

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...

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Problems solved by technology

O-glycosylation occurs on serine or threonine, and there is no characteristic modification sequence to predict the modification site; the structure of O-glycosylation is complex, and there are at least 8 core structures; Generic endonuclease that releases chains from proteins, which makes analysis of O-glycosylation sites difficult

[0004] Usually the analysis of O-glycosylation sites uses chemical release methods such as β-elimination methods (ZhengY, et al. Talanta2009, 78:358-363), but chemical methods are sometimes difficult to control and cause some side reactions

In addition to the above methods, there are other exonuclease methods ( Petal.JProteomeRes, 2007, 6:3021-3031), the identified O-glycosylation sites are relatively limited

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