Comprehensive Profiling of N‑Linked Glycosylation Sites in HeLa Cells Using Hydrazide Enrichment

The adenocarcinoma cell line HeLa serves as a model system
for
cancer research in general and cervical cancer in particular. In this
study, hydrazide enrichment in combination with state-of-the art nanoLC–MS/MS
analysis was used to profile N-linked glycosites in HeLa cells. N-Linked
glycoproteins were selectively enriched in HeLa cells by the hydrazide
capture method, which isolates all glycoproteins independent of their
glycans. Nonglycosylated proteins were removed by extensive washing.
N-Linked glycoproteins were identified with the specific NXT/S motif
and deamidated asparagine (N). Deglycosylation was carried out in
both H216O and H218O to
confirm the deamidation. NanoLC–MS/MS analysis indicated that
the method selectively enriched at least 100 fold N-linked glycosites
in HeLa cells. When both the membrane and cytosolic fractions were
used, a total of 268 unique N-glycosylation sites were identified
corresponding to 106 glycoproteins. Bioinformatic analysis revealed
that most of the glycoproteins identified are known to have an impact
on cancer and have been proposed as biomarkers.