Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry
Comprehensive identification of post-translational modifications of rat bone osteopontin by mass spectrometry
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DOI：
10.1021/bi050109p
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发表时间：
2005-05-10
期刊：
影响因子：
2.9
通讯作者：
Lajoie, G
中科院分区：
文献类型：
--
作者：
Keykhosravani, M;Doherty-Kirby, A;Lajoie, G
骨桥蛋白(OPN)是一种高度修饰的蛋白质，存在于许多组织中，与多种生理和病理过程有关。骨桥蛋白是羟基磷灰石晶体形成的有效抑制物，并刺激破骨细胞的骨吸收；这些活性以及其他活性依赖于蛋白质的磷酸化。我们用质谱仪对从大鼠骨中提纯的OPN的翻译后修饰进行了全面的分析。基质辅助激光解吸飞行时间(MALDI-TOF)MS显示酶脱磷前后的质量分别为37.6 kDa和36.8 kDa，相当于约10.4个磷酸基团的含量。利用蛋白水解酶和串联MS，我们定位了29个磷酸化位点：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和S297。此外，Y150被证明是硫酸盐化的，而T107、TI10、T116和T121是O-糖基化的。在潜在的N-糖基化位点没有检测到糖链。还存在其他修饰，包括脱酰胺化、氧化和氨基甲酰化。即使在唾液酸酶处理之后，67-102残基的36个氨基酸序列也不能被详细分析，这可能是因为存在大量的酸性残基。与之前描述的牛乳异构体相比，大鼠骨OPN是硫酸盐化的，具有额外的糖基化位点，许多不同的磷酸化位点，以及较低的总磷酸盐含量。
Osteopontin (OPN) is a highly modified protein that is found in many tissues and has been associated with a variety of physiological and pathological processes. Bone OPN is a potent inhibitor of hydroxyapatite crystal formation and stimulates bone resorption by osteoclasts; these activities, as well as others, are dependent upon phosphorylation of the protein. We have used mass spectrometry (MS) to perform 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 kDa before and after enzymatic dephosphorylation, respectively, corresponding to a content of approximately 10.4 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, Y150 was shown to be sulfated and T 107, T I 10, T 116, and T 121 are O-glycosylated. No glycan was detected at the potential N-glycosylation site. Other modifications, including deamidation, oxidation, and carbamylation, are also present. A 36-amino acid sequence from residues 67-102 could not be analyzed in detail, even after sialidase treatment, presumably because of the presence of a large number of acidic residues. In comparison to the previously characterized cow milk isoform, rat bone OPN is sulfated and has an additional site of glycosylation, many different sites of phosphorylation, and a lower overall phosphate content.