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Complementary Application of LC-ESI-TOF-MS and LC-ICP-MS for the Analysis of Native and Recombinant Metalloproteins

Gunda Koellensperger¹, Stephan Hann¹, Christian Obinger¹, Hanspeter Rottensteiner², Simon Daubert², Gerhard Stingeder¹

Correspondence address: Gunda Koellensperger, University of Natural Resources and Applied Life Sciences-BOKU-Vienna, Chemistry, Muthgasse 18, Vienna, A-1190 Austria.

Keywords: Mass Accuracy; Mass Spectrometry, Inductively Coupled Plasma; Mass Spectrometry, Time of Flight; Metalloprotein.

Novel aspect: Elemental mass spectrometry in bioinorganic studies. Determination of oxidation state of metal center in proteins by LC-ESI-TOF MS.

 

This study addresses the complementary use of LC-ICP-MS and LC-ESI-MS for characterization of metalloproteins. The fundamental experiments on isolated metalloproteins show the complexity of measuring metal-protein association in biological samples. These basic studies form in our opinion an important starting point for the ultimate goal of comprehensive metalloproteins mapping. A set of recombinant and native Copper proteins was investigated. Moreover, putative zinc proteins, which are supposed to play a central role in the protein import mechanism into peroxisomes were characterized. SEC-ICP-MS and IC-ICP-MS were evaluated for quantification of metal to sulfur ratios. As already described elsewhere ¹ quantification of sulfur in metalloproteins offers an alternative for protein quantification. As the amino acid sequence is known, and hence the amount of sulfur containing amino acids, the sulfur content gives an accurate value of the molar protein concentration. Consequently, the metal/sulfur ratio characterizes the stoichiometric metal content of the protein. For recombinant proteins the knowledge of metal integration addresses quality control since in most instances heterologous expression of metalloproteins results in the apoform and needs the design of reconstitution protocols. In the case of putative metalloproteins the investigation of metal binding is a prerequisite for studying fundamental structure-function relationships.

The studied set of proteins made clear that the benefits of hyphenated ICP-MS analysis for sulfur/metal determination could only be fully exploited provided that the separation methods were thoroughly validated for each metalloprotein. Complementary determination of molar mass of the apoproteins by LC-ESI-TOF-MS was necessary as the loss of the N-terminus or point mutations occurring during heterologous expression changed the sulfur content of the protein. Excellent precision ranging at 3 ppm (N=5) could be achieved by TOF-MS for multiple charged ions of the investigated proteins. The precision of the molar mass determination ranged at 5-15 ppm (N=5). The intact metalloproteins were measured by flow injection - ESI-TOFMS under non-denaturing conditions (pH 5). Stoichiometry of metal ligation and even the oxidation state of the metal center could be assessed for two copper recombinant proteins.

1. S. Hann, G. Koellensperger, C. Obinger, P. Furtmueller and G. Stingeder, J. Anal. Atom. Spectrom. 19, 74 (2004).