Choice of mobile phase: Implications for size exclusion chromatography-inductively coupled plasma-mass spectrometry analyses of copper, zinc and iron metalloproteins

Abstract

The correct identification of the metalloproteins present in human tissues and fluids is essential to our understanding of the cellular mechanisms underpinning a host of health disorders. Separation and analysis of biological samples are typically done via size exclusion chromatography hyphenated with inductively coupled plasma-mass spectrometry (SEC-ICP-MS). Although this technique can be extremely effective in identification of potential metalloproteins, the choice of mobile phase may have a marked effect on results, results by adversely affecting metal-protein bonds of the metalloproteins of interest. To assess the choice of mobile phase on SEC-ICP-MS resolution and the resulting metalloproteome pattern, we analysed several different sample types (brain homogenate; Cu/Zn-superoxide dismutase (SOD1); a molecular weight standard mix containing ferritin (Ft), ceruloplasmin (Cp), cytochrome c (CytC), vitamin B12 (B12) and thyroglobulin (Tg) using six different mobile phase conditions (200 mM, pH 7.5 solutions of ammonium salts nitrate, acetate, and sulfate; HEPES, MOPS and Tris-HCl). Our findings suggest that ammonium nitrate, ammonium acetate and Tris-HCl are optimal choices for the mobile phase, with the specific choice being dependent on both the number of samples and method of detection that is hyphenated with separation. Furthermore, we found that MOPS, HEPES and ammonium sulfate mobile phases all caused significant changes to peak resolution, retention time and overall profile shape. MOPS and HEPES, in particular, produced additional Fe peaks that were not detected with any of the other mobile phases that were investigated. As well as this, MOPS and HEPES both caused significant concentration dependent matrix suppression of the internal standard.