title: High-Throughput Analysis of IgG Fc Glycopeptides by LC-MS.
abstract: This chapter contains a nanoscale liquid chromatography-mass spectrometry method for the glycoform profiling of the conserved Fc N-glycosylation site of monoclonal and polyclonal immunoglobulin G (IgG). It describes in detail LaCyTools, a program for automated data (pre-)processing of the obtained LC-MS data. The minimal sample preparation necessary is explained as well as an optional method for affinity purification of (polyclonal) antibodies from serum or plasma.After (optional) affinity purification, the pure IgG is cleaved with trypsin. The tryptic glycopeptides are separated almost exclusively on their peptide backbone. This ensures similar response factors for all glycoforms in the MS detection and allows the collection of separate glycoform profiles for different IgG isoforms or allotypes. LaCyTools automatically performs label-free (relative) quantitation of the obtained data after minimal manual input and additionally calculates several quality criteria which can be used for data curation at the level of both individual analytes and entire LC-MS runs.
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Falck
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands.
authors.forename: Bas C
authors.initials: BC
authors.lastname: Jansen
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands.
authors.forename: Noortje
authors.initials: N
authors.lastname: de Haan
authors.affiliation: Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333, ZA, Leiden, The Netherlands. m.wuhrer@lumc.nl.|Division of BioAnalytical Chemistry, VU University Amsterdam, De Boelelaan 1083, 1081, HV, Amsterdam, The Netherlands. m.wuhrer@lumc.nl.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods*; D054794:Glycomics / Q000379:methods*; D006020:Glycopeptides / Q000032:analysis*; D006031:Glycosylation; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D011489:Protein Denaturation; D012984:Software
chemical_list: D006020:Glycopeptides; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G
keywords: Automated data analysis; Bottom-up proteomics; Glycoform profiling; Glycopeptide analysis; Glycoproteomics; High-throughput glycosylation profiling; Immunoglobulin G; Label-free quantification; Liquid chromatography–mass spectrometry (LC-MS); Quality scores