Water is thought to play a dominant role in protein folding, yet gaseous multiply protonated proteins from which the water has been completely removed show hydrogen/deuterium (H/D) exchange behavior similar to that used to identify conformations in solution. Indicative of the gas-phase accessibility to D20, multiply-charged (6+ to 17+) cytochrome c cations exchange at six (or more) distinct levels of 64 to 173 out of 198 exchangeable H atoms, with the 132 H level found at charge values 8+ to 17+. Infrared laser heating and fast collisions can apparently induce ions to unfold to exchange at a higher distinct level, while chargestripping ions to lower charge values yields apparent folding as well as unfolding.Water is thought to be a key factor in the spontaneous folding of a protein into its bioactive conformation (1-5); "water interactions are of the essence in the function of real-life protein molecules" (6). However, a preliminary report (7) of the research described here gave hydrogen/deuterium (H/D) exchange evidence of at least three conformations of waterfree cytochrome c cations in the gas phase. These multiply protonated species were formed by electrospray ionization, introduced into the high-vacuum region of the Fouriertransform mass spectrometer, and allowed to exchange with D20 at 10-7 Torr (1 Torr = 133.3 Pa). In solution, the degree and sites of H/D exchange are well-established indicators of steric inaccessibility (8) and, thus, of protein conformation. To measure H/D exchange rates, these solution studies have used nuclear magnetic resonance (3, 9, 10), neutron diffraction (11), and mass spectrometry (MS), with MS employed to measure solution-phase H/D exchange rates (12-16), transient intermediates (17), and gaseous noncovalent complexes (18-21).For multiprotonated cytochrome c in the gas phase (7), H/D exchange exhibited pseudo-first-order kinetics for 7+ to 15+ ions. Three distinct exchange levels were dominant, with some charge values exhibiting two of three levels, but the level of highest exchange occurred at an intermediate charge value. Although in solution nonionized cytochrome c mimics this behavior, with the native structure first denatured and then reorganized into the molten globule state with increasing HI concentration (4, 5, 22), the importance of water for in vivo folding appears well established: "the likelihood of a protein refolding in the gas phase is exceedingly small" (23). We report here more definitive high-resolution MS data showing that multiply protonated equine cytochrome c ions undergo H/D exchange with D20 at six distinct levels, with conversion between these levels (presumably unfolding and folding) effected by infrared laser heating and high-velocity collisions or by reducing the number of charges on the ion. MATERIALS AND METHODSSolutions of 20 ,uM equine cytochrome c (Sigma) were electrosprayed [6+ to 9+ ions in pure aqueous solution and 10+ to 17+ ions in methanol/water/acetic acid, 76:22:2 (vol/vol)], and the resulting ions were transported by three rf-...

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A novel surface design compatible with the open cell geometry allows nonglancing angle collisions of selected ions stored in a Fourier transform mass spectrometer. Dissociation efficiencies of 36%, 22%, and 14% are achieved for gramicidin S, melittin, and carbonic anhydrase (29 kDa), respectively. Ion neutralization by the surface, which is highly competitive for many singly-charged ions, is minimal, and dissociation products of hypervalent neutral species are not detected. Instead, the spectra are similar to those from collisionally activated and infrared multiphoton dissociation; the fragmentation pathways are relatively independent of the method of energy deposition. For carbonic anhydrase, however, the single event excitation inherent to surface-induced dissociation appears to minimize secondary fragmentation, a critical advantage for tandem mass spectrometry of such large ions. Electrically floating the open cell below ground greatly enhances the collection efficiency.

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For high‐resolution Fourier transform mass spectrometry of electrosprayed proteins, the signal‐to‐noise ratio of measuring nozzle–skimmer fragment ions can be improved substantially by their broadband quadrupolar axialization (QA), even without increasing their concentration in the ion cyclotron resonance cell. Axialization of the product ions makes possible larger, more concentric ion orbits for measurements. QA allowed the identification of new sequence‐indicative product ions from a 29 kDa protein. However, QA of large molecular ions gives little increase in signal, consistent with original trapping near the cell axis. By recentering of ions undergoing ion–molecule reactions, these can be carried out at much higher kinetic energy and pressures; for cytochrome cthis increases the achievable H–D exchange by 40%, corresponding to exchange at all the active sites of its completely denatured conformer.