Method for stabilizing protein-ligand complexes in nanoelectrospray ionization mass spectrometry

The interaction between the bovine pancrease trypsin ( Tryp) and its competitive inhibitor benzamidine ( 1), in solution and the gas phase, is investigated using nanoflow electrospray ionization (nanoES) and Fourier transform ion cyclotron resonance mass spectrometry. In a recent study ( Clark, S. M.; Konermann L. Anal. Chem. 2004, 76, 7077-7083), it was reported that the ( Tryp + 1) complex could not be detected by ES-MS. Here, it is shown that, with gentle sampling conditions, it is possible to detect gaseous protonated ions of the ( Tryp + 1) complex with nanoES-MS. However, the relative abundance of the detected ( Tryp + 1)(n+) ions is lower than expected, based on solution composition, which suggests that dissociation of ( Tryp + 1)(n+) ions occurs during MS sampling. The dissociation pathways and corresponding Arrhenius parameters for the protonated ( Tryp + 1)(n+) ions, at n = 7- 9, are determined from time-resolved thermal dissociation experiments, implemented with the blackbody infrared radiative dissociation technique. The gaseous ( Tryp + 1)(n+) ions are found to have short lifetimes, e. g., < 0.6 s, at temperatures of > 100 degrees C. The use of solution additives, including polyols, carbohydrates, amino acids, and small organic molecules, to stabilize the ( Tryp + 1)(n+) ions during nanoES-MS analysis is investigated. Notably, the addition of imidazole to the nanoES solution is shown to preserve the ( Tryp + 1)(n+) ions. The K-assoc value, (1.9 +/- 0.2) x 10(4) M-1, determined for the ( Tryp + 1) complex by the direct ES-MS method is in agreement with values determined by other analytical methods. The stabilizing effect of imidazole in nanoES-MS is further demonstrated for the interaction between carbonic anhydrase II and 5-(dimethylamino) naphthalene-1- sulfonamide. The stabilizing effect of imidazole may be due to enhanced evaporative cooling achieved by the dissociation of molecules of imidazole, bound nonspecifically, from the protein-ligand complex in the ion source.