Secondary and tertiary structures of gaseous protein ions characterized by electron capture dissociation mass spectrometry and photofragment spectroscopy

Abstract

Over the last decade a variety of MS measurements, such as H͞D exchange, collision cross sections, and electron capture dissociation (ECD), have been used to characterize protein folding in the gas phase, in the absence of solvent. To the extensive data already available on ubiquitin, here photofragmentation of its ECDreduced (M ؉ nH) (n؊1)؉• ions shows that only the 6؉ to 9؉, not the 10؉ to 13؉ ions, have tertiary noncovalent bonding; this is indicated as hydrogen bonding by the 3,050 -3,775 cm ؊1 photofragment spectrum. ECD spectra and H͞D exchange of the 13؉ ions are consistent with an all ␣-helical secondary structure, with the 11؉ and 10؉ ions sufficiently destabilized to denature small bend regions near the helix termini. In the 8؉ and 9؉ ions these terminal helical regions are folded over to be antiparallel and noncovalently bonded to part of the central helix, whereas this overlap is extended in the 7؉, 6؉, and, presumably, 5؉ ions to form a highly stable three-helix bundle. Thermal denaturing of the 7؉ to 9؉ conformers both peels and slides back the outer helices from the central one, but for the 6؉ conformer, this instead extends the protein ends away to shrink the three-helix bundle. Thus removal of H 2O from a native protein negates hydrophobic interactions, preferentially stabilizes the ␣-helical secondary structure with direct solvation of additional protons, and increases tertiary interhelix dipole-dipole and hydrogen bonding.

Radical-driven dissociation of odd-electron peptide radical ions produced in 157 nm photodissociation

“…Gas-phase reactions of multiply-charged protein and polypeptide ions with electrons or negatively-charged organic ions (electron capture dissociation, ECD or electron-transfer dissociation, ETD, respectively) yield abundant protein radical ions without changing protein conformation [11,12,21]. Although the majority of these hydrogen-rich radical ions readily dissociate, charge-reduced radical ions have also been abundantly observed [22,23].…”

Electron-Transfer Ion/Ion Reactions of Doubly Protonated Peptides: Effect of Elevated Bath Gas Temperature

“…This interpretation is consistent with evidence that suggests that it is the sites of charge solvation that determine where cleavage is observed in ECD, with structural heterogeneity determining how widely varied these sites are. [27][28][29][30][31] In this work, we examine the ETD behavior of cations derived from 27 peptide species, 22 of which were derived from tryptic digestion, that range in size from 5 to 18 residues in a 3D quadrupole ion trap, both at room temperature and at an elevated bath gas temperature. Of particular interest is both the overall efficiency of ETD, in terms of parent ions converted to product ions, and the sequence coverage represented by the observed ETD fragments.…”

Charge-state resolved mid-infrared spectroscopy of a gas-phase protein

“…Gas-phase techniques that have been implemented to study protein conformations include ion mobility, 10 H/D exchange 11 and protein fragmentation 12,13 experiments. Using those techniques, effects of, for example, the solvent (e.g.…”

“…IR spectra in the hydrogen stretching region around 3300 cm À1 have recently been obtained for the protein ubiquitin. 13 Here, we show the IR spectra for several charge states of the protein bovine heart cytochrome c, which is, to our knowledge, the largest molecule ever investigated with mid-IR spectroscopy in the gas phase.…”