Site-specific structural characterization of O-glycosylation and identification of phosphorylation sites of recombinant osteopontin

• Published in: Biochimica et biophysica acta Vol. 1854; no. 6; pp. 581 - 591
• Main Authors: Li, Hong, Shen, Huali, Yan, Guoquan, Zhang, Yang, Liu, Mingqi, Fang, Pan, Yu, Hongxiu, Yang, Pengyuan
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2015
• Subjects: Chromatography, Liquid - methods; Glycosylation; HEK293 Cells; Humans; Mass spectrometry; Mass Spectrometry - methods; O-glycosylation; Osteopontin; Osteopontin - chemistry; Osteopontin - metabolism; Peptide Mapping - methods; Phosphorylation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Site-specific; Site-specific; PNA; DBA; Phosphorylation; RCA120; PTM; PHA-E; MSn; MAL-II; O-glycosylation; OPN; MS/MS; BSA; SD; RPLC; SBA; GSL-I; Fuc; HexNAc; CKII; MSA; GlcNAc; PSA; CID; aa; UEA-I; MALDI-TOF; MS; MGCK; Con A; PMF; Osteopontin; Lac/LacNAc; CIP; LCA; AAL; O-GlcNAc; HEK; GalNAc; WGA; MMP; GRIP; Gal; SNA-I; Hex; NeuAc; Mass spectrometry
• ISSN: 1570-9639; 0006-3002; 1878-1454
• DOI: 10.1016/j.bbapap.2014.09.025

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-Lex/a antigens [Neuα2-3Galβ1-4(Fucα1-3)GlcNAc/Neuα2-3Galβ1-3(Fucα1-4)GlcNAc]. MS3 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. •Site-specific O-glycosylation of recombinant human osteopontin (OPN) was analyzed.•28 O-glycopeptides carrying highly heterogeneous O-glycans were detected.•Some important carbohydrate epitopes were revealed.•26 phosphorylation sites were identified, including a novel one (Y209).