FAIMS analysis of bacterial glycopeptides

Hydrophilic Interaction Liquid Chromatography (HILIC) glycopeptide enrichment is an indispensable tool for the high-throughput characterisation of glycoproteomes. Despite its utility HILIC enrichment is associated with a number of short comings such as requiring large amounts of starting material, potentially introducing chemical artefacts such as formylation, and biasing/under-sampling specific classes of glycopeptides. Here we investigate HILIC enrichment independent approaches for the study of bacterial glycoproteomes. Using three Burkholderia species (B. cenocepacia; B. dolosa and B. ubonensis) we demonstrate that short aliphatic O-linked glycopeptides are typically absent from HILIC enrichments yet are readily identified in whole proteome samples. Using FAIMS fractionation we show at low compensation voltages (CVs) short aliphatic glycopeptides can be enriched from complex samples providing an alternative means to identify glycopeptides recalcitrant to hydrophilic based enrichment. Combining whole proteome and FAIMS analysis we show the observable glycoproteome of these Burkholderia species is at least 30% larger than initially thought. Excitingly the ability to enrich glycopeptides using FAIMS appears generally applicable with N-linked glycopeptides of Campylobacter fetus subsp. fetus also enrichable at low FAIMS CVs. Combined these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.