Chemical Deamidation: A Common Pitfall in Large-Scale N-Linked Glycoproteomic Mass Spectrometry-Based Analyses

N-Linked glycoproteins are involved in several diseases
and are
important as potential diagnostic molecules for biomarker discovery.
Therefore, it is important to provide sensitive and reliable analytical
methods to identify not only the glycoproteins but also the sites
of glycosylation. Recently, numerous strategies to identify N-linked
glycosylation sites have been described. These strategies have been
applied to cell lines and several tissues with the aim of identifying
many hundreds (or thousands) of glycosylation events. With high-throughput
strategies however, there is always the potential for false positives.
The confusion arises since the protein N-glycosidase F (PNGase F)
reaction used to separate N-glycans from formerly glycosylated peptides
catalyzes the cleavage and deamidates the asparagine residue. This
is typically viewed as beneficial since it acts to highlight the modification
site. We have evaluated this common large-scale N-linked glycoproteomic
strategy and proved potential pitfalls using Escherichia coli as a model organism, since it lacks the N-glycosylation machinery
found in mammalian systems and some pathogenic microbes. After isolation
and proteolytic digestion of E. coli membrane proteins,
we investigated the presence of deamidated asparagines. The results
show the presence of deamidated asparagines especially with close
proximity to a glycine residue or other small amino acid, as previously
described for spontaneous in vivo deamidation. Moreover, we have identified
deamidated peptides with incorporation of 18O, showing
the pitfalls of glycosylation site assignment based on deamidation
of asparagine induced by PNGase F in 18O-water in large-scale
analyses. These data experimentally prove the need for more caution
in assigning glycosylation sites and “new” N-linked
consensus sites based on common N-linked glycoproteomics strategies
without proper control experiments. Besides showing the spontaneous
deamidation, we provide alternative methods for validation that should
be used in such experiments.