title: Simple capillary electrophoresis-mass spectrometry method for complex glycan analysis using a flow-through microvial interface.
abstract: A flow-through microvial is used to interface capillary electrophoresis and mass spectrometry (CE-MS) to develop a method for simultaneous profiling both neutral and sialylated glycans without derivatization or labeling. The CE separation was performed at near-zero electroosmotic flow in a capillary with neutral, hydrophilic coating, using 50 mM ammonium acetate in 20% methanol (pH 3.1) as the background electrolyte. The method was optimized with reversed CE polarity and negative ion ESI-MS. Enzymatically released N-glycans from human immunoglobulin G (IgG) were used as the test sample. The approach was also used to study the more complex N-glycans from recombinant human erythropoietin (rHuEPO) expressed in Chinese hamster ovary (CHO) cells. Glycoscreening of rHuEPO was performed using a triple quadrupole MS and an ultrahigh resolution TOF-MS. The high sensitivity and high mass accuracy of the TOF-MS revealed the presence of more than 70 glycans. Three mono- and di-sialylated tetra-antennary N-glycans and one mono-sialylated tri-antennary N-glycan of rHuEPO are reported for the first time. Further glycan heterogeneity was identified of the highly sialylated N-glycans of rHuEPO by extensive acetylation, Neu5Ac/Neu5Gc variation and the presence of N-acetyl-lactosamine repeats. For comparative purposes, porous graphitic carbon-based LC-MS/MS was also used to glycoprofile rHuEPO. This work demonstrates the potential of CE-MS to provide a comprehensive glycosylation profile with detailed features of the secondary glycan modifications. The CE-MS based method eliminates the need to label the N-glycans, as well as the requirement to desialylate before analysis, and could complement other established techniques for glycan characterization of therapeutic glycoproteins.
authors.affiliation: Department of Chemistry, University of British Columbia , Vancouver V6T 1Z4, British Columbia, Canada.
authors.forename: Roxana G
authors.initials: RG
authors.lastname: Jayo
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.forename: Petrus W
authors.initials: PW
authors.lastname: Lindenburg
authors.forename: Rob
authors.initials: R
authors.lastname: Haselberg
authors.forename: Thomas
authors.initials: T
authors.lastname: Hankemeier
authors.forename: Rawi
authors.initials: R
authors.lastname: Ramautar
authors.forename: David D Y
authors.initials: DD
authors.lastname: Chen
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods*; D004921:Erythropoietin / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis*; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
chemical_list: C554270:EPO protein, human; D006023:Glycoproteins; D007074:Immunoglobulin G; D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin