Proteomics Analysis of O‑GalNAc Glycosylation in Human Serum by an Integrated Strategy

The
diversity of O-linked glycan structures has drawn increasing
attention due to its vital biological roles. However, intact O-glycopeptides
with different glycans are typically not well elucidated using the
current methods. In this work, an integrated strategy was developed
for comprehensive analysis of O-GalNAc glycosylation by combining
hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type
collision induced decomposition (beam-CID) detection, and in silico
deglycosylation method for spectra interpretation. In this strategy,
the intact O-GalNAc glycopeptides were selectively enriched and the
original spectra obtained by time-of-flight (TOF)-CID were preprocessed
using an in silico deglycosylation method, enabling direct searching
without setting multiple glycosylation modifications, which could
significantly decrease the search space. This strategy was applied
to analyze the O-GalNAc glycoproteome of human serum, leading to identification
of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About
81% of the glycopeptides contained at least one sialic acid, which
could reveal the microheterogeneity of O-GalNAc glycosylation. Up
until now, this is the largest data set of intact O-GalNAc glycoforms
from complex biological samples at the proteome level. Furthermore,
this method is readily applicable to study O-glycoform heterogeneity
in other complex biological systems.