title: Targeted, Site-specific quantitation of N- and O-glycopeptides using18O-labeling and product ion based mass spectrometry.
abstract: The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and18O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.
authors.affiliation: Glycomics and Glycoproteomics & Biologics Characterization Facility, Centre for Cellular and Molecular Platforms, National Centre for Biological Sciences (NCBS) - TIFR, GKVK Post, Bellary Road, Bangalore, Karnataka, 560065, India.
authors.forename: Jandhyam
authors.initials: J
authors.lastname: Srikanth
authors.affiliation: Glycomics and Glycoproteomics & Biologics Characterization Facility, Centre for Cellular and Molecular Platforms, National Centre for Biological Sciences (NCBS) - TIFR, GKVK Post, Bellary Road, Bangalore, Karnataka, 560065, India.
authors.forename: Rathinasamy
authors.initials: R
authors.lastname: Agalyadevi
authors.affiliation: Glycomics and Glycoproteomics & Biologics Characterization Facility, Centre for Cellular and Molecular Platforms, National Centre for Biological Sciences (NCBS) - TIFR, GKVK Post, Bellary Road, Bangalore, Karnataka, 560065, India. babup@ncbs.res.in.
authors.forename: Ponnusamy
authors.initials: P
authors.lastname: Babu
mesh_terms: D000068800:Etanercept / Q000737:chemistry; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D010103:Oxygen Isotopes / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D000069283:Rituximab / Q000737:chemistry
chemical_list: D006020:Glycopeptides; D010103:Oxygen Isotopes; D000069283:Rituximab; D000068800:Etanercept
keywords: 18O–labeling and biotherapeutics; Glycopeptide; Mass spectrometry; Quantitative glycoproteomics