S67: Defining the Metalloproteomes of Microorganisms

One cannot predict from genome sequences either the metals that a microorganism assimilates or the number and types of metal-binding proteins that the cell contains. We recently developed a generally-applicable experimental approach to determine all metals utilized by a microorganism and to identify its metalloproteins on a genome-wide scale. The method combines multi-level liquid chromatography, high-throughput tandem mass spectrometry (HT-MS/MS) and a comprehensive 53 elemental analysis using inductively coupled plasma mass spectrometry (ICP-MS). This approach shifts the focus from the purification of proteins to the purification of metal peaks in order to determine which proteins they are associated with. In the pilot study, we characterized cytoplasmic metalloproteins from an exemplary microorganism. Of 343 metal peaks in chromatography fractions, 158 did not match any predicted metalloprotein. 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. Since it is impossible to purify every specific metalloprotein found in the cytoplasm of the organism, we have developed a computational data infrastructure coupled with statistical analyses to facilitate the identification, pursuit and purification of novel metalloproteins. In addition, HPLC/ICP-MS techniques are being developed to assist in elucidating metal-protein interactions by the analyses of co-purification of metals and proteins across a large fractionation space. The integration of experimental and computational approaches provides a high throughput platform that promises to expand our understanding of the role of metals in cell biology, microbial growth and mechanisms of toxicity.