title: Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.
abstract: Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.
authors.affiliation: Institute of Microbiology, Department of Biology, ETH Zurich , Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Chia-Wei
authors.initials: CW
authors.lastname: Lin
authors.affiliation: LimmatTech Biologics AG, CH-8952 Schlieren, Switzerland.
authors.forename: Micha A
authors.initials: MA
authors.lastname: Haeuptle
authors.affiliation: Institute of Microbiology, Department of Biology, ETH Zurich , Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Aebi
mesh_terms: D001592:Benzyl Alcohols / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D006020:Glycopeptides / Q000032:analysis*; D008970:Molecular Weight; D036103:Nanotechnology; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D001592:Benzyl Alcohols; D006020:Glycopeptides; C046886:3-nitrobenzyl alcohol