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 <i>Escherichia coli</i> 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 <i>E. coli</i> 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