Broadband quadrupolar axialization of large multiply charged ions to enhance measurement and minimize conformational restrictions.

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 c this increases the achievable H-D exchange by 40%, corresponding to exchange at all the active sites of its completely denatured conformer.

Gas-phase folding and unfolding of cytochrome c cations.

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 D2O, 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 charge-stripping ions to lower charge values yields apparent folding as well as unfolding.

Surface-induced dissociation of multiply-protonated proteins.

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.