Comprehensive Identification of Post-translational Modifications of Rat Bone Osteopontin by Mass Spectrometry

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• 作者：Mandana Keykhosravani ; Amanda Doherty-Kirby ; Cunjie Zhang ; Dyanne Brewer ; Harvey A. Goldberg ; Graeme K. Hunter ; and Gilles Lajoie
• 刊名：Biochemistry
• 出版年：2005
• 出版时间：May 10, 2005
• 年：2005
• 卷：44
• 期：18
• 页码：6990 - 7003
• 全文大小：334K
• 年卷期：v.44,no.18(May 10, 2005)
• ISSN：1520-4995

文摘

Osteopontin (OPN) is a highly modified protein that is found in many tissues and has beenassociated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor ofhydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well asothers, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) toperform a comprehensive analysis of the post-translational modification of OPN purified from rat bone.Matrix-assisted laser desorption time-of-flight (MALDI-TOF) MS showed masses of 37.6 and 36.8 kDabefore and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately10.4 phosphate groups. Using proteolytic digestion and tandem MS, we localized 29 sites of phosphorylation: S10, S11, S46, S47, T50, S60, S62, S65, S146, T154, S160, S164, S167, S193, S196, S203,S220, S223, S232, S241, S245, S257, S262, S267, S278, S290, S295, S296, and S297. In addition, Y150was shown to be sulfated and T107, T110, T116, and T121 are O-glycosylated. No glycan was detectedat the potential N-glycosylation site. Other modifications, including deamidation, oxidation, andcarbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzedin detail, even after sialidase treatment, presumably because of the presence of a large number of acidicresidues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated andhas an additional site of glycosylation, many different sites of phosphorylation, and a lower overallphosphate content.