Zabrouskov Vlad, Ge Ying, Schwartz Jae, Walker Jeffery W
Thermo Fisher Scientific, San Jose, California 95134, USA.
Mol Cell Proteomics. 2008 Oct;7(10):1838-49. doi: 10.1074/mcp.M700524-MCP200. Epub 2008 Apr 28.
Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI approximately 9.5) contains twelve serine, eight threonine, and three tyrosine residues, which presents a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we used top down electron capture dissociation and electron transfer dissociation MS to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty-six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. Electron capture dissociation of monophosphorylated cTnI localized two major basal phosphorylation sites: a well known site at Ser(22) and a novel site at Ser(76)/Thr(77), each with partial occupancy (Ser(22): 53%; Ser(76)/Thr(77): 36%). Top down MS(3) analysis of diphosphorylated cTnI revealed occupancy of Ser(23) only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser(22/23) pair. Top down MS of cTnI provides unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing establishment of the relevance of such modifications to physiological functions and disease status.
心肌肌钙蛋白I（cTnI）是细肌丝肌钙蛋白 - 原肌球蛋白调节复合物的抑制亚基，在心动周期中对心肌舒张是必需的。cTnI仅在心肌中表达，在临床上广泛用作心脏损伤的血清生物标志物。cTnI的体内功能受磷酸化和蛋白水解作用影响；因此，对完整蛋白质翻译后修饰的分析应极大地有助于理解心脏调节机制，并可能改善cTnI作为疾病生物标志物的性能。cTnI（24 kDa，pI约为9.5）含有12个丝氨酸、8个苏氨酸和3个酪氨酸残基，这对明确鉴定磷酸化位点和定量位置异构体提出了挑战。在本研究中，我们使用自上而下的电子捕获解离和电子转移解离质谱来揭示从人心脏组织中纯化的cTnI的分子复杂性。人cTnI的高分辨率质谱图显示出高度的异质性，对应于磷酸化、乙酰化、氧化和C末端蛋白水解。尽管在SDS - PAGE上呈单一清晰条带，但在单个ESI/FTMS谱图中检测到36个cTnI的分子离子。单磷酸化cTnI的电子捕获解离定位了两个主要的基础磷酸化位点：一个是位于Ser(22)的已知位点，另一个是位于Ser(76)/Thr(77)的新位点，每个位点都有部分占有率（Ser(22)：53%；Ser(76)/Thr(77)：36%）。双磷酸化cTnI的自上而下MS(3)分析显示，仅在双磷酸化物种中Ser(23)被占据，这与Ser(22/23)对的顺序（或有序）磷酸化/去磷酸化一致。cTnI的自上而下质谱为揭示其分子复杂性和定量磷酸化位置异构体提供了独特的机会，从而能够确定这些修饰与生理功能和疾病状态的相关性。
