The titration of ras-GDP with Mg2+ also demonstrates that PLIMSTEX is applicable to those relatively weak noncovalent protein-ligand complexes that may be difficult to detect by direct electrospray. Although ESI-MS can detect the 20 mM of ras-GDP binary complex and its nonspecific sodium adducts in 2 mM ammonium acetate, pH 5.2 , the ternary complex of ras-GDP-Mg2+ does not survive under similar conditions because the constants (Ka) for Mg2+ binding are relatively small. Even with ras-GDP, a relatively strong complex, the solvent pH and electrospray source conditions are critical in a direct ESI-MS analysis. In the PLIMSTEX protocol, the ligands release upon quenching at low pH to form ras-GDP and allow an easy detection of apo-ras under normal ESI-MS conditions (47.5:47.5:5.0 of CH3CN:H2O:CH3COOH, pH 2.6). PLIMSTEX does not rely on the ability of MS to measure solution concentration from peak intensities but rather to measure m/z. The two complexes are distinguishable by their different H/D exchange kinetics, leading to different deuterium uptakes at certain exchange times.
Was this article helpful?