What Are We Missing by Using Hydrophilic Enrichment? Improving Bacterial Glycoproteome Coverage Using Total Proteome and FAIMS Analyses

Hydrophilic interaction liquid chromatography
(HILIC) glycopeptide
enrichment is an indispensable tool for the high-throughput characterization
of glycoproteomes. Despite its utility, HILIC enrichment is associated
with a number of shortcomings, including requiring large amounts of
starting materials, potentially introducing chemical artifacts such
as formylation when high concentrations of formic acid are used, and
biasing/undersampling specific classes of glycopeptides. Here, we
investigate HILIC enrichment-independent approaches for the study
of bacterial glycoproteomes. Using three Burkholderia species (Burkholderia cenocepacia, Burkholderia Dolosa, and Burkholderia ubonensis), we demonstrate that short
aliphatic O-linked glycopeptides are typically absent from HILIC enrichments,
yet are readily identified in whole proteome samples. Using high-field
asymmetric waveform ion mobility spectrometry (FAIMS) fractionation,
we show that at high compensation voltages (CVs), short aliphatic
glycopeptides can be enriched from complex samples, providing an alternative
means to identify glycopeptide recalcitrant to hydrophilic-based enrichment.
Combining whole proteome and FAIMS analyses, we show that the observable
glycoproteome of these Burkholderia species is at least 25% larger than what was initially thought.
Excitingly, the ability to enrich glycopeptides using FAIMS appears
generally applicable, with the N-linked glycopeptides of Campylobacter fetus subsp. fetus also being enrichable at high FAIMS CVs. Taken together, these results
demonstrate that FAIMS provides an alternative means to access glycopeptides
and is a valuable tool for glycoproteomic analysis.