Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry
Identification of reactive cysteines in a protein using arsenic labeling and collision-induced dissociation tandem mass spectrometry
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DOI：
10.1021/pr700662y
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Le, X. Chris
中科院分区：
文献类型：
--
作者：
Lu, Meiling;Wang, Hailin;Le, X. Chris
三价砷对蛋白质中的巯基（如游离半胱氨酸）具有高亲和力。在这里，我们描述了使用这个属性开发的碰撞诱导解离（CID）串联质谱（MS/MS）技术用于识别蛋白质中的反应性半胱氨酸。一种三价砷物种，二甲基亚胂酸（DMA（III）），与残基质量（103.9607）和质量缺陷不同于正常的20个氨基酸，被用来选择性地标记蛋白质中的反应性半胱氨酸残基。砷标记序列的CID碎片离子偏离了更丰富的正常碎片，否则这些碎片将与感兴趣的离子重叠。沿着内部和亚铵离子，砷标记的碎片离子作为MS/MS特征，用于识别结合位点和评估蛋白质中单个半胱氨酸残基的相对反应性。使用这种方法，我们已经确定了两个高度反应性的结合位点在大鼠血红蛋白（Hb）：Cys-13 α和Cys-125 β。Cys-13 α与喂砷大鼠Hb中的DMA（III）结合，这种结合是导致大鼠血液中砷蓄积的原因，而Cys-125 β与大鼠血液中的谷胱甘肽结合。这项研究揭示了大鼠Hb中半胱氨酸的相对反应性，按以下降序排列：Cys-13 alpha >> Cys-111 alpha > Cys-104 alpha和Cys-13 alpha >> Cys-125 β> Cys-93 β。砷标记是简单和快速的活性结合位点的鉴定，而无需酶消化和酸水解，并用于表征和鉴定其他蛋白质中的金属结合位点。
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-13 alpha and Cys-125 beta. Cys-13 alpha 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-125 beta 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-13 alpha >> Cys-111 alpha > Cys-104 alpha and Cys-13 alpha >> Cys-125 beta > Cys-93 beta. 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.