N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry
In: Journal of proteome research, Jg. 13 (2014-02-18), Heft 3
Online
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Zugriff:
N-Glycosylation site analysis of baker's yeast Saccharomyces cerevisiae is of fundamental significance to elucidate the molecular mechanism of human congenital disorders of glycosylation (CDG). Here we present a mass spectrometry (MS)-based workflow for the profiling of N-glycosylated sites in S. cerevisiae proteins. In this workflow, proteolytic glycopeptides were enriched by using a hydrophilic material named Click TE-Cys to improve the glycopeptide selectivity and coverage. To enhance the reliability of the identified results, the enriched glycopeptides were subjected to parallel deglycosylation by using two endoglycosidases (i.e., PNGase F and Endo Hf), respectively, prior to LC-MS/MS analysis. On the basis of the workflow, a total of 135 N-glycosylated sites including 6 known, 93 potential, and 36 novel sites were identified and mapped to 79 proteins. Among the novel-type sites, nine sites from eight proteins, which were simultaneously identified via PNGase F and Endo Hf deglycosylation, are believed to possess high confidence. The established workflow, together with the profile of N-glycosylated sites, will contribute to the improvement of S. cerevisiae model for revealing the pathogenesis of CDG.
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N-Glycosylation site analysis of proteins from Saccharomyces cerevisiae by using hydrophilic interaction liquid chromatography-based enrichment, parallel deglycosylation, and mass spectrometry
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Autor/in / Beteiligte Person: | Guo, Zhimou ; Cao, Liwei ; Guo, Yunü ; Shen, Aijin ; Yu, Long ; Liang, Xinmiao |
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Zeitschrift: | Journal of proteome research, Jg. 13 (2014-02-18), Heft 3 |
Veröffentlichung: | 2014 |
Medientyp: | unknown |
ISSN: | 1535-3907 (print) |
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