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Method for analyzing protein O-glycosylation sites

A technology for glycosylation sites and proteins, which is applied in the field of analyzing protein O-glycosylation sites, can solve the problems of undiscovered, difficult to control chemical methods, and difficult to analyze O-glycosylation sites, and achieves a simple method. Effect

Active Publication Date: 2014-12-10
SHANDONG UNIV
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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 (Zheng Y, 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 ( P et al.J Proteome Res, 2007, 6:3021-3031), the identified O-glycosylation sites are relatively limited

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  • Method for analyzing protein O-glycosylation sites

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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...

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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.

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...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06G01N30/72G01N30/89
Inventor 迟连利白雪李道远
Owner SHANDONG UNIV
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