Journal
JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY
Volume 21, Issue 11, Pages 1224-1231Publisher
ROYAL SOC CHEMISTRY
DOI: 10.1039/b604974p
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The complementary use of LC-ESI- MS and LC-ICP- MS for characterization of native and recombinant copper proteins ( molar mass range 10 - 20 kDa) was investigated. SEC and IC separation protocols were implemented for hyphenated ICP- MS analysis. The studies showed that validation of the methods addressing metal integration on a quantitative basis via metal to sulfur ratios demanded complementary determinations of molar mass. Reversed phase LC- ESI- TOF- MS measurements showed point mutation for the investigated recombinant apo- plastocyanin. Moreover, both recombinant proteins, i. e. plastocyanin and the CuA domain of cytochrome c oxidase from the cyanobacterium Synechocystis, lost their N- terminal amino acid upon expression. Since in both cases methionine formed the N- terminus the theoretical metal to sulfur ratio of the protein was changed. Excellent precision ranging at 3 ppm ( N = 5) could be achieved for the determination of multiply charged ion patterns by LC- ESI- TOF- MS. The precision of the molar mass determination after deconvolution ranged at 5 - 15 ppm ( N = 5). The intact metal containing copper proteins were measured by flow injection- ESI- TOF- MS under non-denaturing conditions (pH 5). Mass accuracy of ESI- TOF- MS allowed confirming not only stoichiometry of metal ligation but also the oxidation state of the metal center in plastocyanin and the CuA domain of cytochrome c oxidase. Moreover, IC-ICP-MS measurements on isotopically enriched Cu proteins were accomplished showing the potential of hyphenated ICP- MS analysis in future tracer studies.
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