Equilibrium unfolding of proteins monitored by electrospray ionization mass spectrometry: distinguishing two-state from multi-state transitions.

The acetic acid-induced unfolding of cytochrome c (cyt c) and apomyoglobin (aMb) are studied under equilibrium conditions by electrospray ionization (ESI) mass spectrometry (MS). The folding states of the proteins in solution are monitored by the charge state distributions that they produce during ESI. A tightly folded protein shows lower charge states than the same protein in an unfolded conformation. The ESI-MS data presented in this study show that during the denaturation of cyt c, only two distinct charge state distributions are observed. These can be attributed to the native and to the acid-unfolded conformation, respectively. In the transition region where the folded and the unfolded conformation are both present in solution, these two distributions are observed simultaneously, thus giving rise to a bimodal ESI mass spectrum. These data reflect a highly cooperative (two state) folding behavior. In contrast, the acid-induced unfolding of aMb is accompanied by gradual shifts in the maxima of the observed charge state distribution. This indicates a non-cooperative unfolding behavior involving multiple protein conformations. The observations made here suggest that ESI-MS might be a general method for assessing the cooperativity of protein unfolding transitions. This study also addresses the issue of 'secondary' solvent effects for ESI-MS studies on the acid-induced unfolding of proteins. These effects influence the ESI charge state distribution without being related to conformational changes of the protein in solution and could potentially complicate the interpretation of ESI mass spectra. Data obtained for bovine pancreatic trypsin inhibitor and ubiquitin indicate that secondary solvent effects influence the observed charge state distributions only to a very minor extent between pH 8.5 and 2.5.