Using Laser Ablation Inductively Coupled-Mass Spectrometry to Detect Nickel-Containing Proteins.

Benjamin A. Neely^1,4, William C. Davis^2,4, David Point^2,4, Joy D. Van Nostrand^1,4, Steven J. Christopher^1,2,4 Paul M. Bertsch^1,3, and Pamela J. Morris^1,4

¹ Marine Biomedicine and Environmental Sciences Center, Medical University of South Carolina, Charleston, SC

² U.S. National Institute of Standards and Technology, Hollings Marine Laboratory, Charleston, SC

³ Savannah River Ecology Laboratory, University of Georgia, Aiken, SC

⁴ Hollings Marine Laboratory, Charleston, SC

Metallomics, the identification and/or quantification of elemental species, is an emerging field that attempts to identify the distribution, coordination environment and concentrations of elemental species in biological samples.  A new technology in metallomics uses routine high-resolution protein separation by gel electrophoresis and laser ablation inductively coupled plasma-mass spectrometry (GE-LA-ICP-MS).  This approach allows for a fast, robust method to screen and quantify metal abundance directly from a gel.  It can also be used synergistically with matrix-assisted laser desorption ionization-time of flight for further identification of metal speciation.  In order to further develop and evaluate GE-LA-ICP-MS, we screened a single protein, Cytochrome C, for metal content as proof of principle.  Cytochrome C is a matrix soluble protein involved in the electron-transport chain and contains a single heme-bound iron (Fe).  A commercially available form of the protein was separated by SDS-PAGE, stained with Coomassie Blue and analyzed by LA-ICP-MS.  Without using collision cell ICP-MS to limit ⁵⁶Fe interferences, there was no detectable ⁵⁷Fe response.  There was a significant detectable response for ⁶³Cu, ⁶⁵Cu, ⁵³Cr, ¹¹²Cd and ⁶⁶Zn that correlated well with the mass of protein present in the gel.  Although these impurities weren’t expected, the ability of the method to detect their levels was proof of the versatility and sensitivity of GE-LA-ICP-MS.  Ongoing studies include using soft techniques for gel separation such as Native-PAGE to maintain loosely bound metal species after separation.  Additionally, GE-LA-ICP-MS is being used to detect nickel (Ni) containing species in a Ni resistant microorganism, Burkholderia cepacia PR1₃₀₁.  By screening Ni exposed cell extracts to identify Ni-binding proteins we can better identify the mechanism of Ni resistance which will contribute to our overall knowledge of how Ni as a contaminant affects microorganisms.