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.

Coexisting stable conformations of gaseous protein ions.

For further insight into the role of solvent in protein conformer stabilization, the structural and dynamic properties of protein ions in vacuo have been probed by hydrogen-deuterium exchange in a Fourier-transform mass spectrometer. Multiply charged ions generated by electrospray ionization of five proteins show exchange reactions with 2H2O at 10(-7) torr (1 torr = 133.3 Pa) exhibiting pseudo-first-order kinetics. Gas-phase compactness of the S-S cross-linked RNase A relative to denatured S-derivatized RNase A is indicated by exchange of 35 and 135 hydrogen atoms, respectively. For pure cytochrome c ions, the existence of at least three distinct gaseous conformers is indicated by the substantially different values--52, 113, and 74--of reactive H atoms; the observation of these same values for ions of a number--2, 7, and 5, respectively--of different charge states indicates conformational insensitivity to coulombic forces. For each of these conformers, the compactness in vacuo indicated by these values corresponds directly to that of a known conformer structure in the solution from which the conformer ions are produced by electrospray. S-derivatized RNase A ions also exist as at least two gaseous conformers exchanging 50-140 H atoms. Gaseous conformer ions are isometrically stable for hours; removal of solvent greatly increases conformational rigidity. More specific ion-molecule reactions could provide further details of conformer structures.

Fourier-transform electrospray instrumentation for tandem high-resolution mass spectrometry of large molecules.

Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York, USA Mass spectrometry instrumentation providing unit resolution and lo-ppm mass accuracy for molecules larger than 10 kDa was first reported in 1991. This instrumentation has now been improved with a 6.2-T magnet replacing that of 2.8 T, a more efficient vacuum system, ion injection with controlled ion kinetic energies, accumulated ion trapping with an open-cylindrical ion cell, acquisition of 2M data points, and updated electrospray apparatus. The resulting capabilities include resolving power of 5 × 10(5) for a 29-kDa protein, less than l-ppm mass measuring error, and dissociation of protein molecular ions to produce dozens of fragment ions whose exact masses can be identified from their mass-to-charge ratio values and isotopic peak spacing.

Negative ion electrospray mass spectrometry of nucleotides: ionization from water solution with SF6 discharge suppression.

The total current and selected ion currents from the electrospray ionization (ES1) of 10(-5) M solutions of cocaine hydrochloride and deoxycytidine monophosphate (dCMP) monosodium salt in methanol and water solvents were compared in positive and negative ion modes, respectively, without and with SF6, gas as a discharge suppressant. The ESI onset voltages (Von), were the same for the positive and negative ion modes. The Von, for methanol was much lower than that for water and in agreement with the equation of D. P. H. Smith, who attributes the difference to the higher surface tension of water. The onset of electric discharge (Vdis) without SF6, occurred at lower capillary voltages for the negative relative to the positive ion mode for methanol; but Vdis is much higher than Von for methanol, and discharges do not interfere with ESI operation. For water, Von ≈ Vdis in the absence of SF6, in the negative ion mode, and ESI operation is impossible without SF6, discharge suppression. The discharge problem in the positive ion mode is less severe, but SF6, is still very useful. A dynamic range of 10 (-7)-10(-5) M was obtained by selected ion monitoring of [dCMP - H](-) at 4.5 and 20 µL/min. flows. Subpicomole detection limits for the nucleotide salt were obtained under these conditions.