title: A method for high-throughput, sensitive analysis of IgG Fc and Fab glycosylation by capillary electrophoresis.
abstract: The N-glycan of the IgG constant region (Fc) plays a central role in tuning and directing multiple antibody functions in vivo, including antibody-dependent cellular cytotoxicity, complement deposition, and the regulation of inflammation, among others. However, traditional methods of N-glycan analysis, including HPLC and mass spectrometry, are technically challenging and ill suited to handle the large numbers of low concentration samples analyzed in clinical or animal studies of the N-glycosylation of polyclonal IgG. Here we describe a capillary electrophoresis-based technique to analyze plasma-derived polyclonal IgG-glycosylation quickly and accurately in a cost-effective, sensitive manner that is well suited for high-throughput analyses. Additionally, because a significant fraction of polyclonal IgG is glycosylated on both Fc and Fab domains, we developed an approach to separate and analyze domain-specific glycosylation in polyclonal human, rhesus and mouse IgGs. Overall, this protocol allows for the rapid, accurate, and sensitive analysis of Fc-specific IgG glycosylation, which is critical for population-level studies of how antibody glycosylation may vary in response to vaccination or infection, and across disease states ranging from autoimmunity to cancer in both clinical and animal studies.
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States.
authors.forename: Alison E
authors.initials: AE
authors.lastname: Mahan
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States.
authors.forename: Jacquelynne
authors.initials: J
authors.lastname: Tedesco
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States.
authors.forename: Kendall
authors.initials: K
authors.lastname: Dionne
authors.affiliation: Department of Biochemistry, Oxford University, Oxford, United Kingdom.
authors.forename: Kavitha
authors.initials: K
authors.lastname: Baruah
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH, United States.
authors.forename: Hao D
authors.initials: HD
authors.lastname: Cheng
authors.affiliation: Department of Neurology, Brigham and Women's Hospital, Boston, MA, United States.
authors.forename: Philip L
authors.initials: PL
authors.lastname: De Jager
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States; Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, MA, United States.
authors.forename: Dan H
authors.initials: DH
authors.lastname: Barouch
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States.
authors.forename: Todd
authors.initials: T
authors.lastname: Suscovich
authors.affiliation: Thayer School of Engineering, Dartmouth College, Hanover, NH, United States.
authors.forename: Margaret
authors.initials: M
authors.lastname: Ackerman
authors.affiliation: Department of Biochemistry, Oxford University, Oxford, United Kingdom.
authors.forename: Max
authors.initials: M
authors.lastname: Crispin
authors.affiliation: Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, United States. Electronic address: galter@partners.org.
authors.forename: Galit
authors.initials: G
authors.lastname: Alter
mesh_terms: D000818:Animals; D019075:Electrophoresis, Capillary / Q000379:methods*; D006031:Glycosylation; D057166:High-Throughput Screening Assays; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000378:metabolism*; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism*; D007074:Immunoglobulin G / Q000032:analysis* / Q000276:immunology; D008253:Macaca mulatta; D051379:Mice; D011134:Polysaccharides / Q000032:analysis; D006023:Glycoproteins
chemical_list: D007140:Immunoglobulin Fab Fragments; D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D011134:Polysaccharides; C046179:glycosylated IgG; D006023:Glycoproteins
keywords: Capillary electrophoresis; Fc separation; Glycan analysis; IgG N-glycosylation