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| - Potential for Using Isotopically AlteredMetalloproteins in Species-Specific IsotopeDilution Analysis of Proteins by HPLC Coupled toInductively Coupled Plasma Mass Spectrometry
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| - The production and evaluation of an isotopically enrichedmetalloprotein standard for use as a calibrant in species-specific isotope dilution analysis by HPLC coupled toinductively coupled plasma mass spectrometry is described. Using a model system involving the copper-containing protein rusticyanin (Rc) from the bacteriumAcido-thiobacillus ferrooxidans, it was possible todemonstrate the analytical conditions that could be usedfor the measurement of metalloproteins by on-line IDMSanalysis. Rc was chosen because it is a well-characterizedprotein with an established amino acid sequence and canbe produced in suitable quantities using a bacterialrecombinant system. Three different forms of the proteinwere studied by organic and inorganic mass spectrometry: the native form of the protein containing a naturalisotopic profile for copper, an isotopically enriched species containing virtually all of its copper as the 65Cuisotope, and the nonmetalated apo form. Incorporationof the copper isotopes into the apo form of the proteinwas determined using a UV−vis spectrophotometric assayand shown to be complete for each of the copper-containing species. The experimental conditions requiredto maintain the conformational form of the protein with anonexchangeable copper center were established using+ve electrospray mass spectrometry. A pH 7.0 buffer wasfound to afford the most appropriate conditions, and thiswas then used with HPLC−ICP-MS to verify the stabilityof the copper center by analysis of mixtures of differentisotopic solutions. No exchange of the enriched copperisotope from Rc with an added naturally abundant inorganic copper cation was observed under a neutral pHenvironment, indicating that species-specific ID-MS analysis of metalloproteins is possible.
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