Charge-Surface Correlation in Electrospray Ionization of Folded and Unfolded Proteins

Abstract

Electrospray-ionization mass spectrometry (ESI-MS) is widely used for protein studies. It has been shown that the extent of protein ionization under nondenaturing conditions correlates well with the solvent-accessible surface area of the tridimensional structure, for either folded monomers or multimeric complexes. The goal of this study was to test whether this relation holds for unfolded proteins as well. In order to overcome the paucity of structural data, the server ProtSA was used to model the conformational ensembles of proteins in the unfolded state and generate estimates of the average solvent accessibility. The results are analyzed along with literature data or original measurements by ESI-MS. It is found that the charge-to-surface relation holds for proteins in the unfolded state, free from solvent effects. A double-log plot is derived, in close agreement with published data for folded proteins. These results suggest that the solvent-accessible surface area is a key factor determining the extent of protein ionization by electrospray, independent of the conformational state. This conclusion helps rationalizing conformational effects in protein ESI-MS. The here reported relation can be used to predict the average solvent accessibility and, hence, the state of folding of unknown proteins from ESI-MS data.

Mass spectrometry‐based methods to study protein architecture and dynamics

“…This observation provides strong evidence that it is the physical dimension of the unstructured protein species (and not the balance of acidic and basic sites) that ultimately dictates the extent of multiple charging in ESI MS and suggests that charge-state distributions of protein ions may be used to evaluate the geometry of non-native protein conformations in solution. A recent analysis of a compilation of ESI MS data for a range of unfolded and intrinsically unstructured proteins did reveal a strong correlation between the calculated surface area in solution and the extent of multiple charging, 20 similar to that established earlier for natively folded protein ions [see Eq. (1)].…”

“…Very similar correlations were obtained in a recent comprehensive analysis of the published data. 20 This charge-surface correlation can be used to provide reasonable estimates of protein surface areas in solution. Although it cannot rival the established techniques in terms of measurement precision, it is very useful for characterizing protein assemblies that are not amenable to analysis using traditional biophysical tools due to their transient nature or heterogeneous character.…”

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

“…It has been previously shown that such an empiric charge-to-mass relation reflects a linear log-log chargeto-surface relation, which has been proven to hold both for folded [41,42] and unfolded [43] proteins. The relation holds when comparing the surface of hydrated proteins or protein complexes with different surface-tomass relation [41].…”

Increasing Protein Charge State When Using Laser Electrospray Mass Spectrometry

“…The Z max and Z min observed in the disulfide-reduced mass spectrum were 25+ and 9+, respectively. The increase in overall ion charge of the protein upon disulfide bond reduction is due to unfolding of the protein, which increases the solvent-accessible surface area, improving the protein ionization efficiency [55]. The increase in ion charge above the number of basic amino acids (18) is presumably due to the protonation of amino acids such as proline (Pro), tryptophan (Trp), and glutamine (Gln) [12,56].…”