title: Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides.
abstract: Glycosylation is a common co- and post-translational protein modification, having a large influence on protein properties like conformation and solubility. Furthermore, glycosylation is an important determinant of efficacy and clearance of biopharmaceuticals such as immunoglobulin G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) shows potential for the site-specific glycosylation analysis of IgG at the glycopeptide level. With this approach, however, important information about glycopeptide sialylation is not duly covered because of in-source and metastable decay of the sialylated species. Here, we present a highly repeatable sialic acid derivatization method to allow subclass-specific MALDI-TOF-MS analysis of tryptic IgG glycopeptides. The method, employing dimethylamidation with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different masses for the functionally divergent α2,3- and α2,6-linked sialic acids. Respective lactonization and dimethylamidation leads to their direct discrimination in MS and importantly, both glycan and peptide moieties reacted in a controlled manner. In addition, stabilization allowed the acquisition of fragmentation spectra informative with respect to glycosylation and peptide sequence. This was in contrast to fragmentation spectra of underivatized samples, which were dominated by sialic acid loss. The method allowed the facile discrimination and relative quantitation of IgG Fc sialylation in therapeutic IgG samples. The method has considerable potential for future site- and sialic acid linkage-specific glycosylation profiling of therapeutic antibodies, as well as for subclass-specific biomarker discovery in clinical IgG samples derived from plasma.
authors.affiliation: †Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands.
authors.forename: Noortje
authors.initials: N
authors.lastname: de Haan
authors.affiliation: †Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands.
authors.forename: Karli R
authors.initials: KR
authors.lastname: Reiding
authors.affiliation: ‡Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Markus
authors.initials: M
authors.lastname: Haberger
authors.affiliation: ‡Pharma Biotech Development Penzberg, Roche Diagnostics GmbH, Penzberg, Germany.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Reusch
authors.affiliation: †Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands.
authors.forename: David
authors.initials: D
authors.lastname: Falck
authors.affiliation: †Leiden University Medical Center, Center for Proteomics and Metabolomics, Leiden, The Netherlands.|§Division of BioAnalytical Chemistry, VU University Amsterdam, Amsterdam, The Netherlands.
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry*; D015203:Reproducibility of Results; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D006020:Glycopeptides; D007074:Immunoglobulin G; D019158:N-Acetylneuraminic Acid