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Issue 1, 2004
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SEC-ICP-DRCMS and SEC-ICP-SFMS for determination of metal–sulfur ratios in metalloproteins

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Abstract

Simultaneous determination of Fe/S and Mn/S ratios on transient signals was performed by size exclusion chromatography, hyphenated to inductively coupled plasma mass spectrometry with dynamic reaction cell technology (SEC-ICP-DRCMS), in order to characterize metalloprotein samples by their metal/sulfur ratio. Oxygen was used as the cell gas. The method eliminates the effect of polyatomic isobaric interferences at m/z = 32 by detecting sulfur as the product oxide ion 32S16O which is less interfered. Using the same reaction gas conditions, Fe and Mn were measured at m/z 54, 56 and 55, respectively. SEC-ICP-DRCMS measurements (injection volume 20 µL, SEC flow 300 µL min−1) resulted in excellent limits of detection (LOD). 4.3, 0.4, 2 and 0.6 ng g−1 were assessed for 32S16O, 55Mn, 54Fe and 56Fe, respectively. Reference measurements were carried out by size exclusion chromatography inductively coupled plasma sector field mass spectrometry (SEC-ICP-SFMS), setting the mass resolution at 4500. LODs of 14, 0.5 and 0.4 ng g−1 were obtained for 32S, 55Mn and 56Fe, respectively. The metal/sulfur ratios of 5 commercially available metalloproteins were determined (myoglobin, haemoglobin, cytochrom c, arginase and Mn superoxide dismutase from E. coli). Two proteins were characterized after in-house heterologous expression in a host organism (Mn superoxide dismutase from Anabaena PCC 7120, catalase-peroxidase from Synechocystis PCC 6803). Different calibrants (i.e., Fe3+, Mn2+, SO42−, methionine, myoglobin) for the assessment of inter-elemental ratios have been employed. It was found that calibration using metalloprotein myoglobin is preferable to inorganic standards in terms of uncertainty of measurement. However, all metal/sulfur ratios of the investigated proteins obtained by the different methods agreed within their total combined uncertainty.

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Publication details

The article was received on 02 Sep 2003, accepted on 02 Dec 2003 and first published on 12 Dec 2003


Article type: Paper
DOI: 10.1039/B310650K
J. Anal. At. Spectrom., 2004,19, 74-79

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    SEC-ICP-DRCMS and SEC-ICP-SFMS for determination of metal–sulfur ratios in metalloproteins

    S. Hann, G. Koellensperger, C. Obinger, P. G. Furtmüller and G. Stingeder, J. Anal. At. Spectrom., 2004, 19, 74
    DOI: 10.1039/B310650K

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