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 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.