Identification of Reactive Cysteines in a Protein Using Arsenic Labeling and Collision-Induced Dissociation Tandem Mass Spectrometry

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• 作者：Meiling Lu ; Hailin Wang ; Zhongwen Wang ; Xing-Fang Li ; X. Chris Le
• 刊名：Journal of Proteome Research
• 出版年：2008
• 出版时间：August 2008
• 年：2008
• 卷：7
• 期：8
• 页码：3080-3090
• 全文大小：386K
• 年卷期：v.7,no.8(August 2008)
• ISSN：1535-3907

文摘

Trivalent arsenicals have high affinity for thiols (such as free cysteines) in proteins. We describe here the use of this property to develop a collision-induced dissociation (CID) tandem mass spectrometry (MS/MS) technique for the identification of reactive cysteines in proteins. A trivalent arsenic species, dimethylarsinous acid (DMA^III), with a residue mass (103.9607) and mass defect distinct from the normal 20 amino acids, was used to selectively label reactive cysteine residues in proteins. The CID fragment ions of the arsenic-labeled sequences shifted away from the more abundant normal fragments that would otherwise overlap with the ions of interest. Along with the internal and immonium ions, the arsenic-labeled fragment ions served as MS/MS signatures for identification of the binding sites and for assessment of the relative reactivity of individual cysteine residues in a protein. Using this method, we have identified two highly reactive binding sites in rat hemoglobin (Hb): Cys-13α and Cys-125β. Cys-13α was bound to DMA^III in the Hb of rats fed with arsenic, and this binding was responsible for arsenic accumulation in rat blood, while Cys-125β was found to bind to glutathione in rat blood. This study revealed the relative reactivity of the cysteines in rat Hb in the following decreasing order: Cys-13α ≫ Cys-111α > Cys-104α and Cys-13α ≫ Cys-125β > Cys-93β. Arsenic-labeling is easy and fast for identification of active binding sites without enzymatic digestion and acid hydrolysis, and useful for characterization and identification of metal binding sites in other proteins.