Glycosylation is increasingly recognised as a common protein modification within 
bacterial proteomes. While great strides have been made in identifying species 
that contain glycosylation systems, our understanding of the proteins and sites 
targeted by these systems is far more limited. Within this work we explore the 
conservation of glycoproteins and glycosylation sites across the 
pan-Burkholderia glycoproteome. Using a multi-protease glycoproteomic approach, 
we generate high-confidence glycoproteomes in two widely utilized B. cenocepacia 
strains, K56-2 and H111. This resource reveals glycosylation occurs exclusively 
at Serine residues and that glycoproteins/glycosylation sites are highly 
conserved across B. cenocepacia isolates. This preference for glycosylation at 
Serine residues is observed across at least 9 Burkholderia glycoproteomes, 
supporting that Serine is the dominant residue targeted by PglL-mediated 
glycosylation across the Burkholderia genus. Combined, this work demonstrates 
that PglL enzymes of the Burkholderia genus are Serine-preferring 
oligosaccharyltransferases that target conserved and shared protein substrates.