Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK.
Anal Bioanal Chem. 2012 Apr;402(10):3371-7. doi: 10.1007/s00216-011-5708-6. Epub 2012 Jan 19.
Trace metals are essential for the growth of marine cyanobacteria, being required for key cellular processes such as photosynthesis and respiration. Despite this, the metalloproteomes of marine cyanobacteria are at present only poorly defined. In this study, we have probed the major cobalt, iron, manganese, and nickel-binding proteins in the marine cyanobacterium Synechococcus sp. WH8102 by using two different fractionation approaches combined with peptide mass fingerprinting. For the identification of intact metalloproteins, multidimensional native chromatography was used to fractionate the proteome, followed by inorganic mass spectrometry to identify metal-enriched fractions. This approach led to the detection of nickel superoxide dismutase together with its predicted cofactor. We also explored the utility of immobilized metal affinity chromatography (IMAC) to isolate subpopulations of proteins that display affinity for a particular metal ion. We conclude that low-resolution 2D liquid chromatography is a viable fractionation technique to correlate relatively low-abundance metal ions with their few cellular destinations (e.g. Ni), but challenges remain for more abundant metals with multiple destinations such as iron. IMAC has been shown as a useful pre-fractionation technique to screen for proteins with metal-binding capacity, and may become a particularly valuable tool for the identification of metal-trafficking proteins.
痕量金属对于海洋蓝藻的生长是必不可少的，它们是光合作用和呼吸等关键细胞过程所必需的。尽管如此，海洋蓝藻的金属蛋白组目前还没有很好的定义。在这项研究中，我们使用两种不同的分级方法结合肽质量指纹图谱，探测了海洋蓝藻聚球藻 sp. WH8102 中的主要钴、铁、锰和镍结合蛋白。为了鉴定完整的金属蛋白，我们使用多维天然色谱法对蛋白质组进行分级，然后用无机质谱法鉴定富含金属的级分。这种方法检测到了镍超氧化物歧化酶及其预测的辅因子。我们还探索了固定化金属亲和色谱（IMAC）的应用，以分离对特定金属离子具有亲和力的蛋白质亚群。我们得出结论，低分辨率二维液相色谱是一种可行的分级技术，可以将相对低丰度的金属离子与其少数几个细胞靶标（如 Ni）相关联，但对于具有多个靶标（如铁）的更丰富的金属，仍然存在挑战。IMAC 已被证明是一种有用的预分级技术，用于筛选具有金属结合能力的蛋白质，并且可能成为鉴定金属转运蛋白的特别有价值的工具。
