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

Abstract

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 including 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 Field Asymmetric Waveform IMS (FAIMS) fractionation we show that 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 that 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 the N-linked glycopeptides of Campylobacter fetus subsp. fetus also enrichable at low FAIMS CVs. Taken together, these results demonstrate that FAIMS provides an alternative means to access glycopeptides and is a valuable tool for glycoproteomic analysis.

The Hitchhiker's guide to glycoproteomics

“…IM-MS is a gas-phase separation method of ions based primarily on their mass and charge but also their size and shape [156]. Given that glycopeptides are usually considerably larger and thus occur in higher charge states than unglycosylated co-eluting peptides, IM-MS provides an opportunity to separate glycopeptides from unglycosylated peptides within an online experiment using field asymmetric ion mobility spectrometry (FAIMS) [157]. While these recent technology developments have not yet found widespread application in glycoproteomics [44], promising data have been published for the characterisation of isolated glycoproteins [158][159][160], where IM-MS has been reported to be able to distinguish sialic acid linkage isomers (α2-3 or α2-6) from otherwise isobaric glycopeptide precursors [161], and to enable characterisation of isomeric glycopeptides where different sites on the same peptide are glycosylated [162].…”

Burkholderia PglL enzymes are serine preferring oligosaccharidetransferases which target conserved proteins across the Burkholderia genus

“…125minute gradients were run for each sample altering the buffer composition from 3% Buffer B to 28% B over 95 minutes, then from 28% B to 40% B over 10 minutes, then from 40% B to 80% B over 7 minutes, the composition was held at 80% B for 3 minutes, and then dropped to 3% B over 1 minutes and held at 3% B for another 9 minutes. The Lumos™ Mass Spectrometer was operated in a stepped FAIMS data-dependent mode at three different FAIMS CVs -25, -45 and -65 as previously described 35 . For each FAIMS CV a single Orbitrap MS scan (350-2000 m/z, maximal injection time of 50 ms, an AGC of maximum of 1*10 6 ions and a resolution of 120k) was acquired every 2 seconds followed by Orbitrap MS/MS HCD scans of precursors (NCE 30%, maximal injection time of 80 ms, an AGC set to a maximum of 1*10 5 ions and a resolution of 15k).…”

“…Recently we began exploring the pan-glycoproteome of Burkholderia species focusing on the conservation of the glycans used for glycosylation 12,16 and the properties of glycopeptides which can influence their enrichment using zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) 35 . These studies have highlighted that the glycoproteomes of Burkholderia species appear larger than initially thought, yet, due to the glycosylation sites associated with these proteins not being defined, this has limited our ability to understand the general trends within site utilisation across the Burkholderia pan-glycoproteome.…”

Evaluation and Optimization of High-Field Asymmetric Waveform Ion Mobility Spectrometry for Multiplexed Quantitative Site-specific N-glycoproteomics

“…In addition to proteomics and phosphoproteomics, where different CVs in an LC-FAIMS-MS measurement have been applied to cover complementary gas-phase fractions, LC-FAIMS-MS has also shown increased power to identify cross-linked peptides and bacterial glycopeptides. 20, 21 Since protein glycosylation in human cells differs significantly from that in bacteria, it is necessary to adapt empirically the optimum CV settings for human N-glycoproteome. Especially, it remains unclear how the various glycan structures influence the FAIMS separation of glycopeptides.…”