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.electrospray ionization ͉ gaseous proteins ͉ mass spectrometry ͉ protein conformations ͉ proteomics

The unfolding enthalpy of the native state of ubiquitin in solution is 5 to 8 times that of its gaseous ions, as determined by electron capture dissociation (ECD) mass spectrometry. Although two-state folding occurs in solution, the three-state gaseous process proposed for this by Clemmer and co-workers based on ion mobility data is supported in general by ECD mass spectra, including relative product yields, distinct Delta H(unfolding) values between states, site-specific melting temperatures, and folding kinetics indicating a cooperative process. ECD also confirms that the 13+ ions represent separate conformers, possibly with side-chain solvated alpha-helical structures. However, the ECD data on the noncovalent bonding in the 5+ to 13+ ions, determined overall in 69 of the 75 interresidue sites, shows that thermal unfolding proceeds via a diversity of intermediates whose conformational characteristics also depend on charge site locations. As occurs with increased acidity in solution, adding 6 protons to the 5+ ions completely destroys their tertiary noncovalent bonding. However, solvation of the newly protonated sites to the backbone instead increases the stability of the secondary structure (possibly an alpha-helix) of these gaseous ions, while in solution these new sites aid denaturation by solvation in the aqueous medium. Extensive ion equilibration can lead to even more compact and diverse conformers. The three-state unfolding of gaseous ubiquitin appears to involve ensembles of individual chain conformations in a "folding funnel" of parallel reaction paths. This also provides a further caution for characterizing solution conformers from their gas-phase behavior.

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 approximately 500-residue, approximately 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 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.