| Abstract
| - Analysis of protein ion charge-state distributions in electrospray ionization (ESI) mass spectra has become anindispensable tool in the studies of protein dynamics.However, applications of this technique have been thusfar limited to detection of large-scale conformationaltransitions, which typically change the extent of multiplecharging in a very significant way. However, more subtleconformational changes often elude detection, since theresulting changes of the extent of multiple charging areoften smaller than the charge-state shifts caused by otherexternal factors. Proton-transfer reactions involving protein ions and residual solvent molecules are the majorextrinsic factors causing changes of charge-state distributions unrelated to conformational transitions. Since theextent of such reactions depends on the amount of varioussolvent components transferred to the ESI interface,profound changes of solvent composition may affectprotein ion charge-state distributions not only by affectingprotein higher order structure in solution but also throughmodulation of the efficiency of proton-transfer reactionsin the gas phase. Here we demonstrate that it is possibleto choose experimental conditions in such a way that theinfluence of gas-phase ion chemistry on protein ioncharge-state distributions is not altered over a wide pHrange. This methodology (gas-phase interference-freeanalysis of protein ion charge-state distributions, orGIFPICS) is sensitive enough to allow detection of pepsininactivation under mildly acidic conditions. Pepsin isactive and tightly folded in its native strongly acidicenvironment. Inactivation of pepsin at mildly acidic pHis not accompanied by global unfolding, as spectroscopicmeasurements suggest the protein remains compact.GIFPICS provides a means to observe this small-scaleconformational transition that does not result in proteinunfolding and may in fact elude detection by traditionalspectroscopic techniques.
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