Hao Gang, Wang Danchen, Gu Jane, Shen Qiuying, Gross Steven S, Wang Yanming
Department of Pharmacology, Weill Medical College of Cornell University, New York, New York, USA.
J Am Soc Mass Spectrom. 2009 Apr;20(4):723-7. doi: 10.1016/j.jasms.2008.12.012. Epub 2008 Dec 30.
Protein citrullination is emerging as an important signaling mechanism that modulates a variety of biological processes. This protein modification constitutes only a 1 Da mass shift, and can be readily confused with other common protein modifications that yield an identical mass shift. In an attempt to develop a robust methodology for detection of protein citrullination sites, we analyzed synthetic citrulline-containing peptides by electrospray ionization tandem mass spectrometry. Collision-induced dissociation (CID) spectra revealed abundant neutral loss of 43 Da from citrullinated peptide precursor ions, which was reconciled by elimination of the HNCO moiety (isocyanic acid) from the citrulline ureido group. The elimination occurs readily in multiple charge states of precursor ions and also in b and y ions. HNCO loss in CID spectra provides a novel diagnostic marker for citrullination, and its utility was demonstrated by the discovery of Arg197 as the specific site of citrullination on nucleophosmin upon peptidylarginine deiminase 4 treatment.
蛋白质瓜氨酸化正成为一种重要的信号传导机制，可调节多种生物过程。这种蛋白质修饰仅导致1 Da的质量变化，很容易与产生相同质量变化的其他常见蛋白质修饰相混淆。为了开发一种可靠的检测蛋白质瓜氨酸化位点的方法，我们通过电喷雾电离串联质谱分析了含合成瓜氨酸的肽段。碰撞诱导解离（CID）光谱显示，瓜氨酸化肽前体离子大量中性丢失43 Da，这是通过从瓜氨酸脲基中消除HNCO部分（异氰酸）来解释的。这种消除在多种电荷状态的前体离子以及b离子和y离子中都很容易发生。CID光谱中的HNCO丢失为瓜氨酸化提供了一种新的诊断标记，通过发现精氨酸脱亚氨酶4处理后核磷蛋白上瓜氨酸化的特定位点Arg197，证明了其效用。
