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.

Automated summary

This study explores alternative approaches to studying bacterial glycoproteomes without relying on HILIC enrichment. By analyzing Burkholderia species, it was found that short aliphatic O-linked glycopeptides are absent in HILIC enrichments but can be identified in whole proteome samples. High-field asymmetric waveform ion mobility spectrometry (FAIMS) fractionation allowed for the enrichment of these glycopeptides from complex samples, expanding the observable glycoproteome of Burkholderia species by at least 25%. Additionally, the applicability of FAIMS for glycopeptide enrichment was demonstrated with N-linked glycopeptides from Campylobacter fetus subsp. fetus.