Assessment of metals in reconstituted metallothioneins by electrospray mass spectrometry

Abstract

A method has been developed that combines electrospray ionization mass spectrometry with pH control to provide analysis of metals in native or reconstituted metallothioneins. These metalloproteins cooperatively bind seven divalent metal ions, most commonly Zn2+ and Cd2+. Since the protein is denatured and metal ions are lost below pH3, the pH of the electrospray solution is critical to successful results. The metal-free apoprotein was detected with its most abundant ions in a charge state of 6+, while the folded metallothionein-metal complexes were observed with lower charge states. The retention of seven metals in the molecular ions detected is consistent with the hypothesis that metallothionein retains its conformation in the gas phase. This mass spectrometric technique can be used to determine rapidly and accurately how many and what cations are incorporated per molecule of protein. Information about molar distributions and estimates of relative abundances of various complexes in the sample can be acquired in a single measurement.