title: Characterization of glycation in an IgG1 by capillary electrophoresis sodium dodecyl sulfate and mass spectrometry.
abstract: We report a case study of characterization of a non-enzymatically glycated IgG1 using reducing capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and mass spectrometry (MS). Glycation was found to occur nonspecifically at multiple sites in both the light and heavy chains. The glycated light and heavy chains result in wider peaks eluting late in the reducing CE-SDS profile; in particular, the glycated light chain behaved as a shoulder peak detected by either ultraviolet (UV) or laser-induced fluorescence (LIF) signals. The glycated species can be enriched by boronate affinity chromatography. Analyzing the enriched samples by reversed phase high-performance liquid chromatography in line with time-of-flight MS (RP-HPLC-TOF/MS) revealed adducts of +162 and +324 Da to both the light and heavy chains, suggesting the presence of multiple glycation sites. Tryptic peptide mapping and tandem mass sequencing were used to identify two glycation sites on each of the light and heavy chains.
authors.affiliation: Department of Pharma Technical Development, Genentech, Oceanside, CA 92056, USA.
authors.forename: Timothy
authors.initials: T
authors.lastname: Kaschak
authors.forename: Daniel
authors.initials: D
authors.lastname: Boyd
authors.forename: Boxu
authors.initials: B
authors.lastname: Yan
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D016466:CHO Cells; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D019075:Electrophoresis, Capillary / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods; D010449:Peptide Mapping / Q000379:methods
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G