Protein glycosylation is an important but poorly understood aspect of bacterial physiology. Over the last decade significant strides have been made in the characterisation of bacterial glycosylation with mass spectrometry (MS) emerging as an indispensable tool. While early studies focused on confirming the presence of glycosylation, little attention has been given to characterising the impacts of these modifications or mapping the sites of glycosylation across bacterial proteomes. Focusing on the O-linked oligosaccharyltransferases (PglL/S/O) within Gram negative pathogens, we have begun to explore the preferences of these enzymes as well as how these modifications impact microbial physiology using MS-based systems biology. Using members of the Burkholderia and Neisseria genera as models our work has shown the glycoproteomes of these species are far larger than once thought, with >100 proteins targeted for O-linked glycosylation in some species. While a significant proportion of the glycoproteome has no known function quantitative proteomics has revealed multiple glycoproteins are extremely sensitive to glycosylation silencing being rapidly degraded in its absence. Surprisingly precise glycosylation site mapping has also revealed unexpected preferences in the sites subjected to glycosylation across bacterial species with even closely related enzymes possessing discrete targeting ranges. Combined these studies highlight how bacterial glycosylation is far more nuanced then once thought.