Gas-phase interference-free analysis of protein ion charge-state distributions: Detection of small-scale conformational transitions accompanying pepsin inactivation

Analysis of protein ion charge-state distributions in electrospray ionization (ESI) mass spectra has become an indispensable tool in the studies of protein dynamics. However, applications of this technique have been thus far limited to detection of large-scale conformational transitions, which typically change the extent of multiple charging in a very significant way. However, more subtle conformational changes often elude detection, since the resulting changes of the extent of multiple charging are often smaller than the charge-state shifts caused by other external factors. Proton-transfer reactions involving protein ions and residual solvent molecules are the major extrinsic factors causing changes of charge-state distributions unrelated to conformational transitions. Since the extent of such reactions depends on the amount of various solvent components transferred to the ESI interface, profound changes of solvent composition may affect protein ion charge-state distributions not only by affecting protein higher order structure in solution but also through modulation of the efficiency of proton-transfer reactions in the gas phase. Here we demonstrate that it is possible to choose experimental conditions in such a way that the influence of gas-phase ion chemistry on protein ion charge-state distributions is not altered over a wide pH range. This methodology (gas-phase interference-free analysis of protein ion charge-state distributions, or GIFPICS) is sensitive enough to allow detection of pepsin inactivation under mildly acidic conditions. Pepsin is active and tightly folded in its native strongly acidic environment. Inactivation of pepsin at mildly acidic pH is not accompanied by global unfolding, as spectroscopic measurements suggest the protein remains compact. GIFPICS provides a means to observe this small-scale conformational transition that does not result in protein unfolding and may in fact elude detection by traditional spectroscopic techniques.