title: Site-specific structural characterization of O-glycosylation and identification of phosphorylation sites of recombinant osteopontin.
abstract: Osteopontin (OPN) plays a key role in multiple physiological and pathological processes such as cytokine production, mineralization, inflammation, immune responses, and tumorigenesis. Post-translational modifications (PTMs) of OPN significantly affect its structure and biological properties; however, site-specific characterization of O-glycosylation in human OPN has not been reported. In this work, we profiled the overall glycan pattern of human recombinant OPN using a lectin array and completed detailed structural analysis of O-glycopeptides by mass spectrometry (MS). We detected 28 O-glycopeptides from 7 O-glycosylation regions of human OPN, occupied by highly heterogeneous O-glycans. These O-glycans carried, in part, functionally relevant epitopes such as T antigens (Galβ1-3GalNAcα1-), sialyl-Tn antigens, sialyl-T antigens, and sialyl-Le(x/a) antigens [Neuα2-3Galβ1-4(Fucα1-3)GlcNAc/Neuα2-3Galβ1-3(Fucα1-4)GlcNAc]. MS(3) spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. Furthermore, 26 phosphorylation sites were identified by reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS), including a novel one (Y209). We provide a detailed, site-specific structural characterization of O-glycosylation and identify the phosphorylation sites of OPN. These data lay the foundation for further research into the role of oligosaccharides and phosphorylation of recombinant human OPN. This article is part of a Special Issue entitled: Medical Proteomics.
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200032, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Hong
authors.initials: H
authors.lastname: Li
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Huali
authors.initials: H
authors.lastname: Shen
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200032, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200032, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Mingqi
authors.initials: M
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200032, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China.
authors.forename: Pan
authors.initials: P
authors.lastname: Fang
authors.affiliation: Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China. Electronic address: hongxiuyu@fudan.edu.cn.
authors.forename: Hongxiu
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department of Chemistry, Fudan University, Shanghai 200032, PR China; Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, PR China. Electronic address: pyyang@fudan.edu.cn.
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D053495:Osteopontin / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping / Q000379:methods*; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D053495:Osteopontin
keywords: Mass spectrometry; O-glycosylation; Osteopontin; Phosphorylation; Site-specific