title: High-Throughput Analysis of the Plasma N-Glycome by UHPLC.
abstract: The understanding of glycosylation alterations in health and disease has evolved significantly and glycans are considered to be relevant biomarker candidates. High-throughput analytical technologies capable of generating high-quality, large-scale glycoprofiling data are in high demand. Here, we describe an automated sample preparation workflow and analysis of N-linked glycans from plasma samples using hydrophilic interaction liquid chromatography with fluorescence detection on an ultrahigh-performance liquid chromatography (UHPLC) instrument. Samples are prepared in 96-well plates and the workflow features rapid glycoprotein denaturation, enzymatic glycan release, glycan purification on solid-supported hydrazide, fluorescent labeling, and post-labeling cleanup with solid-phase extraction. The development of a novel approach for plasma N-glycan analysis and its implementation on a robotic platform significantly reduces the time required for sample preparation and minimizes technical variation. It is anticipated that the developed method will contribute to expanding high-throughput capabilities to analyze protein glycosylation.
authors.affiliation: Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Box 440, 405 30, Gothenburg, Sweden.|NIBRT GlycoScience Group, NIBRT-The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co., Dublin, Ireland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Adamczyk
authors.affiliation: NIBRT GlycoScience Group, NIBRT-The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co., Dublin, Ireland.|AbbVie Inc., North Chicago, IL, USA.
authors.forename: Henning
authors.initials: H
authors.lastname: Stöckmann
authors.affiliation: NIBRT GlycoScience Group, NIBRT-The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co., Dublin, Ireland.
authors.forename: Róisín
authors.initials: R
authors.lastname: O'Flaherty
authors.affiliation: Department of Medical Biochemistry and Cell Biology, Institute of Biomedicine, Sahlgrenska Academy, University of Gothenburg, Box 440, 405 30, Gothenburg, Sweden.
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
authors.affiliation: NIBRT GlycoScience Group, NIBRT-The National Institute for Bioprocessing Research and Training, Fosters Avenue, Mount Merrion, Blackrock, Co., Dublin, Ireland. pauline.rudd@nibrt.ie.
authors.forename: Pauline M
authors.initials: PM
authors.lastname: Rudd
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D054794:Glycomics / Q000295:instrumentation / Q000379:methods*; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000295:instrumentation / Q000379:methods; D006801:Humans; D010949:Plasma / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000097:blood / Q000302:isolation & purification; D011489:Protein Denaturation; D012984:Software; D052616:Solid Phase Extraction / Q000295:instrumentation / Q000379:methods
chemical_list: D006023:Glycoproteins; D011134:Polysaccharides
keywords: Automation; Glycan analysis; High-throughput; N-linked glycosylation; Plasma; Robotics; Ultrahigh-performance liquid chromatography