title: High-Throughput and High-Sensitivity Mass Spectrometry-Based N-Glycomics of Mammalian Cells.
abstract: The current protocols for glycomic analysis of cells often require a large quantity of material (5-20 million cells). In order to analyze the N-glycosylation from small amounts of cells (≤1 million) as obtained from, for example, primary cell lines or cell sorting, and in a higher throughput approach, we set up a robust 96-well format PVDF-membrane based N-glycan release protocol followed by linkage-specific sialic acid stabilization, cleanup, and MALDI-TOF-MS analysis. We further evaluated the influence of PNGase F incubation time on the N-glycan profile.
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Postzone S3, Postbus 9600, 2300, RC, Leiden, The Netherlands. s.holst@lumc.nl.
authors.forename: Stephanie
authors.initials: S
authors.lastname: Holst
authors.affiliation: Department of Surgery, Leiden University Medical Center, Postbus 9600, RC, 2300, Leiden, The Netherlands.
authors.forename: Gabi W
authors.initials: GW
authors.lastname: van Pelt
authors.affiliation: Department of Surgery, Leiden University Medical Center, Postbus 9600, RC, 2300, Leiden, The Netherlands.
authors.forename: Wilma E
authors.initials: WE
authors.lastname: Mesker
authors.affiliation: Department of Surgery, Leiden University Medical Center, Postbus 9600, RC, 2300, Leiden, The Netherlands.
authors.forename: Rob A
authors.initials: RA
authors.lastname: Tollenaar
authors.affiliation: Department of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, The Netherlands.
authors.forename: Ana I
authors.initials: AI
authors.lastname: Belo
authors.affiliation: Department of Molecular Cell Biology and Immunology, VU University Medical Centre, Amsterdam, The Netherlands.
authors.forename: Irma
authors.initials: I
authors.lastname: van Die
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Postzone S3, Postbus 9600, 2300, RC, Leiden, The Netherlands.|University Lille, CNRS, UMR 8576, UGSF, Unité de Glycobiologie Structurale et Fonctionnelle, 59 000, Lille, France.
authors.forename: Yoann
authors.initials: Y
authors.lastname: Rombouts
authors.affiliation: Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, Amsterdam, 1081, HV, The Netherlands.|Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, Leiden, 2333, ZA, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D004951:Esterification; D054794:Glycomics / Q000379:methods*; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000737:chemistry; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D052616:Solid Phase Extraction / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D019158:N-Acetylneuraminic Acid
keywords: Cells; Ethyl esterification; MALDI-TOF-MS; N-glycosylation; PNGase F digestion; PVDF filter plates