title: In-depth analyses of native N-linked glycans facilitated by high-performance anion exchange chromatography-pulsed amperometric detection coupled to mass spectrometry.
abstract: Characterization of glycans present on glycoproteins has become of increasing importance due to their biological implications, such as protein folding, immunogenicity, cell-cell adhesion, clearance, receptor interactions, etc. In this study, the resolving power of high-performance anion exchange chromatography with pulsed amperometric detection (HPAE-PAD) was applied to glycan separations and coupled to mass spectrometry to characterize native glycans released from different glycoproteins. A new, rapid workflow generates glycans from 200 μg of glycoprotein supporting reliable and reproducible annotation by mass spectrometry (MS). With the relatively high flow rate of HPAE-PAD, post-column splitting diverted 60% of the flow to a novel desalter, then to the mass spectrometer. The delay between PAD and MS detectors is consistent, and salt removal after the column supports MS. HPAE resolves sialylated (charged) glycans and their linkage and positional isomers very well; separations of neutral glycans are sufficient for highly reproducible glycoprofiling. Data-dependent MS2in negative mode provides highly informative, mostly C- and Z-type glycosidic and cross-ring fragments, making software-assisted and manual annotation reliable. Fractionation of glycans followed by exoglycosidase digestion confirms MS-based annotations. Combining the isomer resolution of HPAE with MS2permitted thorough N-glycan annotation and led to characterization of 17 new structures from glycoproteins with challenging glycan profiles.
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA. Zoltan.Szabo@thermofisher.com.
authors.forename: Zoltan
authors.initials: Z
authors.lastname: Szabo
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: James R
authors.initials: JR
authors.lastname: Thayer
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: Yury
authors.initials: Y
authors.lastname: Agroskin
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: Shanhua
authors.initials: S
authors.lastname: Lin
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Liu
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: Kannan
authors.initials: K
authors.lastname: Srinivasan
authors.affiliation: ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, CA, 95134, USA.|ThermoFisher Scientific, 2845 Argentia Road, Mississauga, Ontario, L5N 8G6, Canada.
authors.forename: Julian
authors.initials: J
authors.lastname: Saba
authors.affiliation: ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, CA, 95134, USA.
authors.forename: Rosa
authors.initials: R
authors.lastname: Viner
authors.affiliation: ThermoFisher Scientific, 355 River Oaks Parkway, San Jose, CA, 95134, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Huhmer
authors.affiliation: ThermoFisher Scientific, 1214 Oakmead Parkway, Sunnyvale, CA, 94088, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Rohrer
authors.affiliation: Roche Diagnostic GmbH, Nonnenwald 2, 82377, Penzberg, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.affiliation: , c/o, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: Rania
authors.initials: R
authors.lastname: Harfouche
authors.affiliation: ThermoFisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA, 94080, USA.
authors.forename: Shaheer H
authors.initials: SH
authors.lastname: Khan
authors.affiliation: ThermoFisher Scientific, 1228 Titan Way, Sunnyvale, CA, 94088, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Pohl
mesh_terms: D000838:Anions / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*
chemical_list: D000838:Anions; D006023:Glycoproteins; D007074:Immunoglobulin G; D011134:Polysaccharides
keywords: Glycan annotation; High performance anion exchange chromatography-pulsed amperometric detection (HPAE-PAD); Hyphenation; Mass spectrometry; Native N-linked glycans