Electrospray ionization mass spectrometry temperature effects on metal ion: Protein stoichiometries and metal-induced conformational changes in calmodulin

The calcium ion (Ca²⁺) binding stoichiometry required to induce a complete, tertiary conformational change of calmodulin is still disputed. Several studies have indicated that this occurs upon the uptake of only two Ca²⁺; more recent reports, however, indicate that four Ca²⁺ are required. We used electrospray ionization (ESI) mass spectrometry under standard ESI conditions (i.e. high temperature, organic co-solvent) to identify definitively the Ca²⁺ stoichiometry required to induce a conformational change in the protein, and we demonstrate that four Ca²⁺ are needed. We then undertook a comparative study on the Ca²⁺-binding of calmodulin using a lower ESI source temperature. Under these conditions we can detect Ca²⁺-saturated calmodulin at much lower Ca²⁺ to protein molar ratios than those observed using typical ESI conditions. This latter observation more closely reflects the solution-phase conditions that are noted using aqueous solution-based spectroscopies to study protein-metal binding.