Metal displacement and stoichiometry of protein-metal complexes under native conditions using capillary...

Increases in the study of protein-metal complexes, as well as in metal displacement in protein-metal complexes under native conditions for optimum catalytic properties in drug research and catalyst design, demands a separation/detection technology that can accurately measure metal displacement and stoichiometry in protein-metal complexes. Both nuclear magnetic resonance (NMR) and X-ray diffraction techniques have been used for this purpose; however, these techniques lack sensitivity. Electrospray ionization mass spectrometry (ESI-MS) using direct infusion offers higher sensitivity than the former techniques and provides molecular distribution of various protein-metal complexes. However, since protein-metal complexes under native conditions usually are dissolved in salt solutions, their direct ESI-MS analysis requires off-line sample clean-up prior to MS analysis to avoid sample suppression during ESI. Moreover, direct infusion of the salty solution promotes non-specific salt adduct formation by the protein-metal complexes under ESI-MS, which complicates the identification and stoichiometry measurements of the protein-metal complexes. Because of the high mass of protein-metal complexes and lack of sufficient resolution by most mass spectrometers to separate non-specific from specific metal-protein complexes, accurate protein-metal stoichiometry measurements require some form of sample clean up prior to ESI-MS analysis. In this study, we demonstrate that capillary electrophoresis/electrospray ionization in conjunction with a medium-resolution (approximately 10,000) mass spectrometer is an efficient and fast method for the measurement of the stoichiometry of the protein-metal complexes under physiological conditions (pH approximately 7). The metal displacement of Co(2+) to Cd(2+), two metal ions necessary for activation in the monomeric AHL lactonase produced by B. thuringiensis, has been used as a proof of concept.