Investigation of the Gas-Phase Structure of Electrosprayed Proteins Using Ion-Molecule Reactions

Abstract

Proton transfer reactions of ammonia, dimethylamine, diethylamine, and trimethylamine with multiply protonated proteins generated by electrospray ionization (ESI) were examined to probe the relationship between solution and gas-phase protein structure and the relationship with ion-molecule reactivity. The ion-molecule reactions were carried out in an atmospheric pressure capillary inlet/reactor based upon an ESI interface to a quadrupole mass spectrometer. Two types of systems were explored: 0) proteins possessing cysteinecysteine disulfide bonds and the analogous disulfide-reduced proteins, and (2) proteins sprayed from solution compositions where the protein has different conformations. While the cysteine-cysteine disulfide-bound proteins were more reactive than equally charged disulfide-reduced proteins under these conditions, no significant reactivity differences were noted for ions arising from different solution conformations. The effect of inlet/reactor temperature on charge distributions with and without amine reagent was also explored, demonstrating that thermal denaturation of proteins can occur in heated capillary inlets. The results are discussed in the context of recent results indicating the persistence of at least some higher order protein structure in the gas phase. (] Am Soc Mass Spectrom 1994,5,207-220) T he introduction of electrospray ionization (ESI) to mass spectrometry [1] has provided exceptional opportunities for studying macromolecules in the gas phase [2][3][4]. These new capabilities also bring new questions, particularly with regard to gas-phase ion structure and its relationship to struchue in solution. Measurement methods reflecting primary structure of biopolymers (e.g., the sequence for proteins) have been rapidly developed, with molecular weight determinations, peptide mapping, and tandem mass spectrometry (MS/MS) finding immediate applications in structure confirmation [5]. Questions regarding preservation of noncovalent associations in the gas phase, while more difficult to answer than those regarding primary structure, are proving to be experimentally tractable because they also rely upon molecular weight measurements [6][7][8][9][10][11][12][13][14][15][16][17]. Examining the higher order gas-phase structure of proteins by mass spectrometry is particularly difficult [18][19][20] because in most cases the relevant structural differences do not lead to differences in molecular weight, although in some cases arguments can be posed based on the observation of buried solvent molecules in protein crystals and their presence or absence in ESI mass spectra [9]. The strikingly different ESI charge-state distributions observed for different solution conformers of many proteins [10, ".0-27] demonstrate the utility of ESI as a probe of solution conformation, but do not inform as to whether the resulting gas-phase proteins differ in any way other than in charge. Some of the questions which arise include: (1) whether the differences in ESI mass spectra of native and denatured protei...

Practical implications of some recent studies in electrospray ionization fundamentals

“…Because the ESI process occurs at atmospheric pressure, and because large quantities of charged solvent molecules are generated in addition to the charged analyte molecules, the ESI‐MS response can be affected by gas‐phase interactions (Ogorzalek Loo & Smith, 1994; Stephenson & McLuckey, 1996; Kebarle & Peschke, 1999; Amad et al, 2000). These gas‐phase interactions occur after the analytes have been released from solution; thus, gas‐phase effects are essentially the “last word” in the electrospray process.…”

What Protein Charging (and Supercharging) Reveal about the Mechanism of Electrospray Ionization

“…[54]. In contrast, CSDs for proteins undergoing a cooperative folding transition change in a different fashion, typically moving from a single charge envelope to two discrete envelopes or to a bimodal distribution [24,25,65,66]. As the unfolding coordinate is traversed, one envelope increases in abundance as the other decreases.…”

Charging of Proteins in Native Mass Spectrometry

“…The proton-transfer reactivities of protein ions with volatile bases, formed by ESI from solutions in which the proteins are denatured, have been investigated experimentally [14, 18, 51–56] and modeled computationally [14, 19, 26, 41, 47, 56]. Proton-transfer reactions between protein ions and volatile basic molecules show that the apparent gas-phase basicity of high charge state ions is lower than that of low charge state ions [14, 18, 52, 56], and that proton-transfer rates between protein ions and basic molecules depend on temperature [51, 57, 58]. Conformation also affects the proton-transfer reactivity of protein ions [14, 52, 56].…”