Proteome-Wide Analysis of N‑Glycosylation Stoichiometry Using SWATH Technology

N-glycosylation is a crucial post-translational modification (PTM) and plays essential roles in biological processes. Several methods have been developed for the relative quantification of N-glycosylation at the proteome scale. However, the proportion of N-glycosylated forms in a total protein popul...

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Bibliographic Details
Published in:Journal of proteome research 2017-10, Vol.16 (10), p.3830-3840
Main Authors: Yang, Xiangyun, Wang, Zhiyuan, Guo, Lin, Zhu, Zhengjiang, Zhang, Yaoyang
Format: Article
Language:English
Subjects:
Amino Acid Sequence - genetics
Animals
CHO Cells
Cricetulus
Glycosylation
HEK293 Cells
Humans
Peptides - chemistry
Peptides - genetics
Protein Processing, Post-Translational - genetics
Proteome - chemistry
Proteome - genetics
Proteomics
Tandem Mass Spectrometry
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Summary:N-glycosylation is a crucial post-translational modification (PTM) and plays essential roles in biological processes. Several methods have been developed for the relative quantification of N-glycosylation at the proteome scale. However, the proportion of N-glycosylated forms in a total protein population, or the “N-glycosylation stoichiometry”, varies greatly among proteins or cellular states and is frequently missing due to the lack of robust technologies. In the present study, we developed a data-independent acquisition (DIA)-based strategy that enabled the in-depth measurement of N-glycosylation stoichiometry. A spectral library containing 3,509 N-glycosylated peptides and 17,525 fragment ions from human embryonic kidney cells 293 (HEK-293) cells was established from which the stoichiometries of 1,186 N-glycosites were calculated. These stoichiometric values differ greatly among different glycosites, and many glycosites tend to occur with low stoichiometry. We then investigated the N-glycosylation changes induced by tunicamycin in HEK-293 cells and by a temperature shift in Chinese hamster ovary (CHO) cells. Quantifying the proteome, N-glycoproteome, and N-glycosylation stoichiometry demonstrated that the regulation of N-glycosylation is primarily achieved by adjusting the N-glycosylation stoichiometry. In total, the stoichiometries of 2,274 glycosites were determined in the current study. Notably, our approach can be applied to other biological systems and other types of PTMs.
ISSN:1535-3893
1535-3907
DOI:10.1021/acs.jproteome.7b00480