APPLICATION OF ELECTROSPRAY MASS-SPECTROMETRY IN PROBING PROTEIN-PROTEIN AND PROTEIN LIGAND NONCOVALENT INTERACTIONS

A novel mass spectrometry-based methodology using electrospray ionization (ESI) is described for the detection of protein-protein [interferon (IFN)-gamma dimer] and protein-ligand [ ras-guanosine diphosphate (GDP)] noncovalent interactions. The method utilizes ESI from aqueous solution at appropriate pH. The presence of the noncovalent complex of the IFN-gamma dimer was confirmed by the observed average molecular weight of 33,819 Da. The key to the detection of the IFN-gamma dimer is the use of an alkaline solution (pH approximately 9) for sample preparation and for mass spectrometry analysis. The effect of the declustering energy in the region of the ion sampling orifice and focusing quadrupole on the preservation of the gas-phase noncovalent complex (IFN-gamma dimer) was also studied. The effect of the declustering energy on complex dissociation was further extended to probe the noncovalent protein-ligand association of ras-GDP. It was found that little energy is required to dissociate the IFN-gamma dimer, whereas a substantial amount of energy is required to dissociate the gas-phase ras-GDP complex.