Microbial metalloproteomes are largely uncharacterized
Microbial metalloproteomes are largely uncharacterized
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DOI：
10.1038/nature09265
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发表时间：
2010-08-05
期刊：
影响因子：
64.8
通讯作者：
Adams, Michael W. W.
中科院分区：
文献类型：
--
作者：
Cvetkovic, Aleksandar;Menon, Angeli Lal;Adams, Michael W. W.
金属离子辅因子为蛋白质提供几乎无限的催化潜力，使电子转移反应成为可能，并对蛋白质稳定性产生重大影响（1，2）。因此，金属蛋白在大多数生物过程中起着关键作用，包括呼吸（铁和铜），光合作用（锰）和药物代谢（铁）。然而，从基因组序列预测生物体从其环境中吸收的金属或在其金属蛋白质组中使用的金属的数量和类型目前是不可能的，因为金属配位位点是多样的且识别不佳（2-4）。我们在这里提出了一个强大的，基于金属的方法来确定所有的金属生物体同化，并确定其金属蛋白在全基因组范围内。这将焦点从经典的基于蛋白质的纯化转移到通过液相色谱、高通量串联质谱（HT-MS/MS）和电感耦合等离子体质谱（ICP-MS）的基于金属的鉴定和纯化，以表征来自示例性微生物（激烈火球菌）的细胞质金属蛋白。在色谱级分中的343个金属峰中，158个与任何预测的金属蛋白不匹配。未分配的峰包括已知使用的金属（钴、铁、镍、钨和锌; 83个峰）以及认为生物体不能吸收的金属（铅、锰、钼、铀和钒; 75个峰）。158个意想不到的金属峰中的8个的纯化产生了四种新的含镍和含铼的蛋白质，而四种纯化的蛋白质含有亚化学计量的错误掺入的铅和铀。对另外两种微生物（大肠杆菌和硫磺硫化叶菌）的分析显示，对更多意想不到的金属有种属特异性同化作用。因此，金属蛋白质组比以前认识到的更加广泛和多样化，并有望为细胞生物学，微生物生长和毒性机制提供关键见解。
Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability(1,2). Consequently, metalloproteins have key roles in most biological processes, including respiration (iron and copper), photosynthesis (manganese) and drug metabolism (iron). Yet, predicting from genome sequence the numbers and types of metal an organism assimilates from its environment or uses in its metalloproteome is currently impossible because metal coordination sites are diverse and poorly recognized(2-4). We present here a robust, metal-based approach to determine all metals an organism assimilates and identify its metalloproteins on a genome-wide scale. This shifts the focus from classical protein-based purification to metal-based identification and purification by liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and inductively coupled plasma mass spectrometry (ICP-MS) to characterize cytoplasmic metalloproteins from an exemplary microorganism (Pyrococcus furiosus). Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. Unassigned peaks included metals known to be used (cobalt, iron, nickel, tungsten and zinc; 83 peaks) plus metals the organism was not thought to assimilate (lead, manganese, molybdenum, uranium and vanadium; 75 peaks). Purification of eight of 158 unexpected metal peaks yielded four novel nickel- and molybdenum-containing proteins, whereas four purified proteins contained sub-stoichiometric amounts of misincorporated lead and uranium. Analyses of two additional microorganisms (Escherichia coli and Sulfolobus solfataricus) revealed species-specific assimilation of yet more unexpected metals. Metalloproteomes are therefore much more extensive and diverse than previously recognized, and promise to provide key insights for cell biology, microbial growth and toxicity mechanisms.