Decharging of Globular Proteins and Protein Complexes in Electrospray

Abstract

Electrospray ionization mass spectrometry (ESI-MS) is a valuable tool in structural biology for investigating globular proteins and their biomolecular interactions. During the electrospray ionization process, proteins become desolvated and multiply charged, which may influence their structure. Reducing the net charge obtained during the electrospray process may be relevant for studying globular proteins. In this report we demonstrate the effect of a series of inorganic and organic gas-phase bases on the number of charges that proteins and protein complexes attain. Solution additives with very strong gas-phase basicities (GB) were identified among the so-called "proton sponges". The gas-phase proton affinities (PA) of the compounds that were added to the aqueous protein solutions ranged from 700 to 1050 kJ mol(-1). Circular dichroism studies showed that in these solutions the proteins retain their globular structures. The size of the proteins investigated ranged from the 14.3 kDa lysozyme up to the 800 kDa tetradecameric chaperone complex GroEL. Decharging of the proteins in the electrospray process by up to 60 % could be achieved by adding the most basic compounds rather than the more commonly used ammonium acetate additive. This decharging process probably results from proton competition events between the multiply protonated protein ions and the basic additives just prior to the final desolvation. We hypothesize that such globular protein species, which attain relatively few charges during the ionization event, obtain a gas-phase structure that more closely resembles their solution-phase structure. Thus, these basic additives can be useful in the study of the biologically relevant properties of globular proteins by using mass spectrometry.

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

“…As expected, the slope of the line depends on the specific protein. Most notably, the intersection of the GB app fitted line with the line of solvent GB corresponds with remarkable agreement to the experimental main (most abundant) charge state under mild ESI conditions [7,10] (Figure 2 and Figure SI-7). Instead, GB app turns out to be systematically underestimated when the calculation is performed constraining the non-hydrogen atoms in the position of the NMR or X-ray structures (Figure 2).…”

“…The extent of protein ionization has been interpreted in terms of GB also in this case [6][7][8][9]. In particular, the GB app of folded cytochrome c has been calculated from the crystallographic structure, accounting for Coulomb repulsions [9].…”

Do Charge State Signatures Guarantee Protein Conformations?

“…Electrolytes with higher gas-phase basicities will, therefore, remove more charge from the protein [26,27]. A study in which lysozyme, among others, was progressively charge-reduced from 10+ to 3+ showed, by circular dichroism spectroscopy, that there is no loss in secondary structure for any of the charge states [28]. This is in direct contrast to the supercharging experiments, in which it is proposed that the corresponding increase in charge, observed for several proteins, is due in part to unfolding in the final stages of desolvation in the electrospray droplet [24].…”

Influence of coulombic repulsion on the dissociation pathways and energetics of multiprotein complexes in the gas phase

“…Where lower complex charge states were desired in positive or negative mode, imidazole was added to nanoES solutions at a concentration of ϳ1 mM. Imidazole is both a relatively strong acid and base in the gas phase (gas-phase basicity ϭ 217 kcal/mol [30], gas-phase acidity ϭ 343 kcal/mol [31], and can engage in proton transfer reactions with the protonated and deprotonated protein ions in the source, thereby reducing their charge states [32].…”