Do ionic charges in ESI MS provide useful information on macromolecular structure?

Abstract

Multiple charging is an intrinsic feature of electrospray ionization (ESI) of macromolecules. While multiple factors influence the appearance of protein ion charge state distributions in ESI mass spectra, physical dimensions of protein molecules in solution are the major determinants of the extent of multiple charging. This article reviews the information that can be obtained by analyzing ionic charge state distributions in ESI MS, as well as potential pitfalls and limitations of this powerful technique. We also discuss future areas of growth with particular emphasis on applications in structural biology, biotechnology (protein-polymer conjugates), and nanomedicine. (J Am Soc Mass Spectrom 2008, 19, 1239 -1246 [2][3][4] preceded the triumphant entry of this ionization technique into the mainstream of biological mass spectrometry by some time. One particular feature of ESI that seemed to be a major impediment for its application to macromolecules was multiple charging, a phenomenon that seemingly complicated the appearance of mass spectra by crowding them with multiple ion peaks corresponding to the same analyte. Although ionic species carrying more than one elementary charge are not uncommon in mass spectra of macromolecular ions generated by means of fast atom bombardment (FAB) or matrix-assisted laser desorption/ionization (MALDI), they typically account for only a small fraction of the overall signal. Furthermore, typical ionic charge density (defined here as the average number of charges per unit mass) is much lower for the macromolecular ions produced by FAB and MALDI compared with ions generated by ESI. Charge reduction strategies via ion-molecular reactions in the gas phase provided an opportunity to simplify ESI mass spectra of polypeptides by eliminating peaks of multiply charged ions. However, it was the realization that multiple charging is an intrinsic property of ESI process and must be dealt with using mathematical tools, that made it a practical tool for the analysis of macromolecules [5].In retrospect, it seems almost ironic that the multiple charging of macromolecules in ESI MS is now viewed not as a liability, but an extremely valuable asset, which provides a new important dimension to the analysis of biopolymers. Indeed, it was not long after the acceptance of ESI MS as a tool for measuring masses of macromolecular ions that observations were made linking the dramatic changes of ionic charge state distributions to protein denaturation in solution [6,7]. These observations brought about the realization of the potential of ESI MS as a means of probing protein higher order structure and detecting large-scale conformational transitions in solution.Natively folded proteins by definition have stable, compact cores sequestered from the solvent and undergo ESI to produce ions carrying a relatively small number of charges. This is because the compact shape of a tightly folded polypeptide chain in solution does not allow the accommodation of a significant number of protons on their surface upon transit...

Do Charge State Signatures Guarantee Protein Conformations?

“…It is important to point out that, as proposed previously, there is a strong correlation between the solvent accessible surface area and the average charge state determined experimentally [29,30] (Figure 1b). For large protein complexes (Figure 1c), experimental evidence supports a mechanism whereby manipulation of surface charge, by charge reduction [31] or enhancement [32], does not necessarily perturb the protein structure as evidenced for transthyretin [31], stable protein 1 [32], and a protective antigen prechannel complex [33].…”

Mass spectrometry‐based methods to study protein architecture and dynamics

“…The anomalous charge density of the products of dissociation is a clear indication that these species are generated in the gas phase rather than in solution. 14 In general, dissociation of protein assemblies in the gas phase almost always manifests itself in ESI mass spectra by giving rise to ionic species of either atypical composition or charge density. 15 Therefore, in most cases, it is not too difficult to make a clear distinction between the dissociation processes occurring in solution and in the gas phase, and the experimental conditions can be adjusted to minimize or completely eliminate the latter.…”

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

“…Higher numbered charge states, with smaller m/z values, represent a greater surface area than lower numbered series and this can be interpreted as a more open structure. Changes in charge states observed during ESI-MS analysis as a function of metallation may indicate the formation of a structure that is folding around the metalbinding site (24). The m/z spectrum of apo SlyD reveals two different populations of SlyD in solution ( Figures 1 and S2), with most of the protein carrying a higher charge (i.e.…”