Mass spectrometry (MS) has been revolutionized by electrospray ionization (ESI), which is sufficiently “gentle” to introduce nonvolatile biomolecules such as proteins and nucleic acids (RNA or DNA) into the gas phase without breaking covalent bonds. Although in some cases noncovalent bonding can be maintained sufficiently for ESI/MS characterization of the solution structure of large protein complexes and native enzyme/substrate binding, the new gaseous environment can ultimately cause dramatic structural alterations. The temporal (picoseconds to minutes) evolution of native protein structure during and after transfer into the gas phase, as proposed here based on a variety of studies, can involve side-chain collapse, unfolding, and refolding into new, non-native structures. Control of individual experimental factors allows optimization for specific research objectives.

For characterization of sequence and posttranslational modifications, molecular and fragment ion mass data from ionizing and dissociating a protein in the mass spectrometer are far more specific than are masses of peptides from the protein's digestion. We extend the ∼500-residue, ∼50-kilodalton (kD) dissociation limitation of this top-down methodology by using electrospray additives, heated vaporization, and separate noncovalent and covalent bond dissociation. This process can cleave 287 interresidue bonds in the termini of a 1314-residue (144-kD) protein, specify previously unidentified disulfide bonds between 8 of 27 cysteines in a 1714-residue (200-kD) protein, and correct sequence predictions in two proteins, one with 2153 residues (229 kD).

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.