An electrospray-ionization mass spectrometry analysis of the pH-dependent dissociation and denaturation processes of a heterodimeric protein

Electrospray ionization mass spectrometry (ESI-MS) was applied to the analy sis of the dissociation and denaturation processes of a heterodimeric yeast killer toxin SMKT. The two distinct subunits of SMKT noncovalently associa te under acidic conditions, but become dissociated and denatured under neut ral and basic conditions. In order to understand the unique pH-dependent de naturation mechanism of this protein, a pH titration was performed utilizin g ESI-MS. The molecular ions of the heterodimer which possesses the highly ordered structure, were mainly observed below pH 4.6. However, the two subu nits immediately dissociated at this pH. The spectra measured with various settings of the mass spectrometer indirectly demonstrated that the pH-depen dent dissociation occurs in the Liquid phase. The current result as well as the three-dimensional structure of SMKT suggest that the deprotonation of a specific carboxyl group triggers a cooperative dissociation process of th is protein. In conclusion, the pH titration of a protein by ESI-MS is parti cularly effective, when the unfolding process or the biological function of the protein is related to the interaction with other molecules, (J Am Soc Mass Spectrom 2000, 11, 54-61) (C) 2000 American Society for Spectrometry.