Over the last decade a variety of MS measurements, such as H/D 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 ECD-reduced (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 H/D exchange of the 13+ ions are consistent with an all α-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 H₂O from a native protein negates hydrophobic interactions, preferentially stabilizes the α-helical secondary structure with direct solvation of additional protons, and increases tertiary interhelix dipole-dipole and hydrogen bonding.

A mass difference between the measured molecular weight of a protein and that of its DNA-predicted sequence indicates sequence errors and/or posttranslational modifications. In the top-down mass spectrometry approach, the measured molecular ion is dissociated, and these fragment masses are matched against those predicted from the protein sequence to restrict the locations of the errors/modifications. The proportion of the ion9s interresidue bonds that are cleaved determines the specificity of such locations; previously, ubiquitin (76 residues) was the largest for which all such bonds were dissociated. Now, cleavages are achieved for carbonic anhydrase at 250 of the 258 interresidue locations. Cleavages of three spectra would define posttranslational modifications at 235 residues to within one residue. For 24 of the 34 possible phosphorylation sites, the cleavages of one spectrum would delineate exactly all −PO₃H substitutions. This result has been achieved with electron-capture dissociation by minimizing the further cleavage of primary product ions and by denaturing the tertiary noncovalent bonding of the molecular ions under a variety of conditions.

Electron capture dissociation (ECD) MS is proving to be unusually valuable for “top down” protein sequencing and identification/localization of posttranslational modifications, because the ECD product ions can represent cleavages between most of a protein's amino acids. As proposed, this unusual reactivity results from immediate local utilization, before randomization, of much of the relatively large (≈6 eV) energy from the electron reaction with the multiply charged protein ion, minimizing the effect of differences in the backbone bond dissociation energies. However, others conclude that e^–capture produces a labile free radical species for which backbone cleavage is the lowest energy reaction. Supporting the nonergodic mechanism, ECD of ubiquitin (M + 12H)¹²⁺ions also yields thermalized radical (M + 12H)^11+·ions that instead lose H· when activated. Also, the ECD spectrum of ubiquitin (M + 13H)¹³⁺ions is nearly unchanged by heating from 25°C to 125°C, demonstrating that this increase in thermal energy is small compared to the energy driving the reaction. These results support initial capture of the electron in a long-lived high-nRydberg state, followed by internal conversion to the product valence state at an energy well above the dissociation barriers. The instantaneous conformation of the valence state is critical, with the observed products supporting an α-helical structure in which the protonated side chain of each basic residue is intercalated to hydrogen-bond to as many as three amide carbonyls. Activation (e.g., heat, collisions, lowered charge) can disrupt this conformation to allow additional H⁺-side-chain interactions and provide more complete sequence coverage.