Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
10537052 | Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics | 2015 | 11 Pages |
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-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.
Keywords
O-GlcNAcO-GlycosylationMSAPMFDBAMSNCKIIOPNWGASBAGalNAcN-acetylgalactosaminePHA-EAALUEA-IO-linked β-N-acetylglucosamineUlex europaeus agglutinin-INeuAcRPLCPisum sativum agglutininRCA120Dolichos biflorus agglutininPhaseolus vulgaris erythroagglutininHexNAcRicinus communis agglutinin IPNACIDConcanavalin AMALDI-TOFPSACIPPTMFucFucoseGlcNAcHEKMMPBSAMS/MSN-acetylhexosaminebovine serum albuminAleuria aurantia lectinamino acidpeanut agglutininSoybean agglutininLens culinaris agglutininPeptide mass fingerprintCollision-induced dissociationLCAOsteopontinN-acetylneuraminic acidpost-translational modificationstandard deviationCon Amatrix-assisted laser desorption ionization-time of flightSite-specificMass spectrometryTandem mass spectrometryPhosphorylationmatrix metalloproteinaseN-acetylglucosamineHexHexosecasein kinase IIreverse-phase liquid chromatographyhuman embryonic kidneyGalGalactoseGRIPWheat germ agglutinin
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Hong Li, Huali Shen, Guoquan Yan, Yang Zhang, Mingqi Liu, Pan Fang, Hongxiu Yu, Pengyuan Yang,