Exploiting Oligosaccharyltransferase-Positive and -Negative and a Multiprotease Digestion Strategy to Identify Novel Sites Modified by N-Linked Protein Glycosylation.

is a bacterial pathogen encoding a unique N-linked glycosylation () system that mediates attachment of a heptasaccharide to N-sequon-containing membrane proteins by the PglB oligosaccharyltransferase (OST). Many targets of PglB are known, yet only a fraction of sequons are experimentally confirmed, and site occupancy remains elusive. We exploited -positive (wild-type; WT) and -negative (Δ) proteomes to identify potential glycosites. The nonglycosylated forms of known glycopeptides were typically increased in protein normalized abundance in Δ relative to WT and restored by reintroduction (Δ::). Sequon-containing peptide abundances were thus consistent with significant site occupancy in the presence of the OST. Peptides with novel sequons were either unaltered (likely not glycosylated) or showed abundance consistent with known glycopeptides. Topology analysis revealed that unaltered sequons often displayed cytoplasmic localization, despite originating from membrane proteins. Novel glycosites were confirmed using parallel multiprotease digestion, LC-MS/MS, and FAIMS-MS to define the glycoproteomes of WT and Δ::. We identified 142 glycosites, of which 32 were novel, and 83% of sites predicted by proteomics were validated. There are now 166 experimentally verified glycosites and evidence for occupancy or nonoccupancy of 31 additional sites. This study serves as a model for the use of OST-negative cells and proteomics for highlighting novel glycosites and determining occupancy in a range of organisms.