Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry
Probing the stoichiometry and oxidation states of metal centers in iron-sulfur proteins using electrospray FTICR mass spectrometry
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DOI：
10.1021/ac991183e
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发表时间：
2000-04-01
影响因子：
7.4
通讯作者：
Amster, IJ
中科院分区：
文献类型：
--
作者：
Johnson, KA;Verhagen, MFJM;Amster, IJ
用电喷雾电离(ESI)傅里叶变换离子回旋共振质谱仪测定了几种铁硫蛋白质中金属中心的化学计量比和氧化态。在非变性条件下将样品引入ESI源，以观察完整的含金属蛋白质离子。蛋白质离子中金属或金属硫簇的化学计量比和氧化状态可以从质谱图中得到，含单核金属的蛋白质和[4Fe-4S]中心非常稳定，生成的分子离子很少或没有碎裂。含有[2Fe-2S]簇的蛋白质不太稳定，会从分子物种中损失一个或两个硫原子，尽管分子离子比碎片峰更丰富。含[3Fe-4S]的蛋白质是所研究物种中最不稳定的，除了一个代表分子离子的峰外，还产生了与失去一个到四个硫原子相对应的丰富的峰。同位素示踪实验表明，硫的损失来源于[3Fe-4S]中心。获得了负离子模式的质谱图，发现与正离子模式相比，负离子模式产生的[3Fe-4S]离子更加稳定。在变性条件下对相同蛋白质进行ESI分析会产生蛋白质载脂蛋白形式的质谱图。在脱辅基蛋白的质谱图中观察到二硫键，而这在全蛋白中不存在。这些是由于负责结合金属中心的半胱氨酸硫原子的氧化偶联造成的。此外，还发现无机硫化物通过在半胱氨酸残基之间形成硫桥而将自己结合到脱辅基蛋白中。
Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry is used to determine the stoichiometry and oxidation states of the metal centers in several iron-sulfur proteins. Samples are introduced into the ESI source under nondenaturing conditions in order to observe intact metal-containing protein ions. The stoichiometry and oxidation state of the metal or metal-sulfur cluster in the protein ion can be derived from the mass spectrum, Mononuclear metal-containing proteins and [4Fe-4S] centers are very stable and yield the molecular ion with little or no fragmentation. proteins that contain [2Fe-2S] clusters are less stable and yield loss of one or two sulfur atoms from the molecular species, although the molecular ion is more abundant than the fragment peaks. [3Fe-4S]-containing proteins are the least stable of the species investigated, yielding abundant peaks corresponding to the loss of one to four sulfur atoms in addition to a peak representing the molecular ion. Isotope labeling experiments show that the sulfur loss originates from the [3Fe-4S] center. Negative ion mode mass spectra were obtained and found to produce much more stable [3Fe-4S]-containing ions than obtained in positive ion mode. ESI analysis of the same proteins under denaturing conditions yields mass spectra of the apo form of the proteins. Disulfide bonds are observed in the apoprotein mass spectra that are not present in the holoprotein. These result from oxidative coupling of the cysteinyl sulfur atoms that are responsible for binding the metal center. In addition, inorganic sulfide is found to incorporate itself into the apoprotein by forming sulfur bridges between cysteine residues.