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

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.