Plimstex in Biologically Relevant Media and High Ionic Strength

Physiological conditions of cellular solutions often include relatively high ionic strength salt media with nonvolatile buffer and high concentrations of salt, which make difficult the detection of protein and protein-ligand complex by direct ESI or MALDI. Even with low ionic strength and a ''mass spectrometry friendly'' solvent, nonspecific adducts may arise, confusing the stoichiometry and affinity determinations. High sensitivity of mass measurements in PLIMSTEX can be achieved because the pH is decreased to quench the exchange, and metal cations and ligands normally dissociate and are removed by online chromatography prior to MS analysis. Further, all forms of the protein revert back to the apo state, giving minimal signal dispersion and good signal-to-noise ratio. The clean up improves the mass resolving power because metal-ion interference is removed. By maintaining a high D/H ratio in the forward exchange and a high H/D ratio in the back exchange, we find a narrow isotope distribution and concomitant improved mass resolving power. Therefore, PLIMSTEX allows one to explore Ca2+ binding to CaM in not only 15 mM of CaM in low ionic strength media (2 mM NH4OAc) but also under conditions with buffer and high ionic strength (50 mM HEPES or 50 mM HEPES with 100 mM KCl; Fig. 11.6).

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