Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with "Slow-Heating" Tandem Mass Spectrometry Methods
Top-Down Mass Analysis of Protein Tyrosine Nitration: Comparison of Electron Capture Dissociation with "Slow-Heating" Tandem Mass Spectrometry Methods
复制标题
DOI：
10.1021/ac101177r
复制
发表时间：
2010-09-01
影响因子：
7.4
通讯作者：
Cooper, Helen J.
中科院分区：
文献类型：
--
作者：
Mikhailov, Victor A.;Iniesta, Jesus;Cooper, Helen J.
蛋白质中的酪氨酸硝化是一种重要的翻译后修饰（PTM），与各种病理状况有关。当存在多个潜在的硝化位点时，串联质谱（MS/MS）方法提供了精确定位硝基酪氨酸的独特工具。电子捕获离解（ECD）是一种功能强大的质谱/质谱方法，其机制不同于碰撞诱导离解（CID）和红外多光子离解（IRMPD）等“慢热”阈值碎裂方法。通常，ECD提供了更均匀的蛋白质主干切割，并保留了不稳定的PTMs。然而，我们实验室最近的研究表明，双电荷硝化肽的ECD被硝基的大电子亲和力所抑制，而CID效率不受硝化的影响。在这里，我们研究了ECD与CID和IRMPD在商用傅里叶变换离子回旋共振（FT-ICR）质谱仪中对肌红蛋白、溶菌酶和细胞色素c的多电荷完整硝化蛋白离子进行自上而下的MS/MS分析的效率。CID和IRMPD比ECD在硝化位点附近产生更多的裂解。然而，ECD片段的总数大于CID或IRMPD，并且许多ECD片段含有硝化位点。我们得出结论，ECD可以用于硝化蛋白质的自上而下分析，但精确定位硝化位点可能需要“慢热”方法中的任何一种。
Tyrosine nitration in proteins is an important post-translational modification (PTM) linked to various pathological conditions. When multiple potential sites of nitration exist, tandem mass spectrometry (MS/MS) methods provide unique tools to locate the nitro-tyrosine(s) precisely. Electron capture dissociation (ECD) is a powerful MS/MS method, different in its mechanisms to the "slow-heating" threshold fragmentation methods, such as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). Generally, ECD provides more homogeneous cleavage of the protein backbone and preserves labile PTMs. However recent studies in our laboratory demonstrated that ECD of doubly charged nitrated peptides is inhibited by the large electron affinity of the nitro group, while CID efficiency remains unaffected by nitration. Here, we have investigated the efficiency of ECD versus CID and IRMPD for top-down MS/MS analysis of multiply charged intact nitrated protein ions of myoglobin, lysozyme, and cytochrome c in a commercial Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. CID and IRMPD produced more cleavages in the vicinity of the sites of nitration than ECD. However the total number of ECD fragments was greater than those from CID or IRMPD, and many ECD fragments contained the site(s) of nitration. We conclude that ECD can be used in the top-down analysis of nitrated proteins, but precise localization of the sites of nitration may require either of the "slow-heating" methods.