Metal ion cofactors afford proteins virtually unlimited catalytic potential, enable electron transfer reactions and have a great impact on protein stability1,2. 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 recognized2,3,4. 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.
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This research is part of the MAGGIE (Molecular Assemblies, Genes and Genomes Integrated Efficiently) project supported by Department of Energy grant (DE-FG0207ER64326). We thank S. Hammond, L. Wells, R. Hopkins and D. Phillips for help with in-gel MS analyses.
The authors declare no competing financial interests.
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Cvetkovic, A., Menon, A., Thorgersen, M. et al. Microbial metalloproteomes are largely uncharacterized. Nature 466, 779–782 (2010). https://doi.org/10.1038/nature09265
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