Charge-State-Dependent Variation of Signal Intensity Ratio between Unbound Protein and Protein–Ligand Complex in Electrospray Ionization Mass Spectrometry: The Role of Solvent-Accessible Surface Area

Abstract

Native electrospray ionization mass spectrometry (ESI-MS) is nowadays widely used for the direct and sensitive determination of protein complex stoichiometry and binding affinity constants ( K). A common yet poorly understood phenomenon in native ESI-MS is the difference between the charge-state distributions (CSDs) of the bound protein-ligand complex (PL) and unbound protein (P) signals. This phenomenon is typically attributed to experimental artifacts such as nonspecific binding or in-source dissociation and is considered highly undesirable, because the determined K values display strong variation with charge state. This situation raises serious concerns regarding the reliability of ESI-MS for the analysis of protein complexes. Here we demonstrate that, contrary to the common belief, the CSD difference between P and PL ions can occur without any loss of complex integrity, simply due to a change in the solvent-accessible surface area (ΔSASA) of the protein upon ligand binding in solution. The experimental CSD shifts for PL and P ions in ESI-MS are explained in relation to the magnitude of ΔSASA for diverse protein-ligand systems using a simple model based on the charged residue mechanism. Our analysis shows that the revealed ΔSASA factor should be considered rather general and be given attention for the correct spectral interpretation of protein complexes.

Mechanistic Insights of IscU Conformation Regulation for Fe–S Cluster Biogenesis Revealed by Variable-Temperature Electrospray Ionization Native Ion Mobility Mass Spectrometry

“…26,28,30 Higher charge states are often observed for unfolded conformations of proteins due to the greater solvent-accessible surface area. [40][41][42] Consistent with this idea, the Zavg increases for both apo-IscU and Zn-IscU at lower and higher temperatures matching previous reports of cold-and heat-induced protein denaturation (Figure 1C). 27,[29][30]43 The addition of ~0.66 equivalents of IscS (IscU:IscS molar ratio of 3:2) does not affect the charge state distributions of apo-IscU and Zn-IscU, suggesting IscS does not significantly alter the overall conformation of IscU.…”

The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor

“…1 and relating these numbers to the known empirical relationship between the average ionic charge ( Z av. ) and the solvent-exposed surface area of the corresponding species in solution (S) [ 30 , 31 ]: where A is a constant and the empirical value of α is 0.69 ± 0.02. Although the surface area values are usually calculated based on the protein crystal structure, using the published crystal structures of the SARS-CoV-2 S-protein to calculate the surface areas of S1 monomers, dimers, and trimers by simply truncating the protein sequence and removing irrelevant segments is likely to generate misleading results for two reasons.…”

Insight into molecular characteristics of SARS-CoV-2 spike protein following D614G point mutation, a molecular dynamics study

“…During the analysis, desired structures were parameterized with AMBER03 force field (Zhang et al, 2019) and simulation was executed for 100 ns. All structural parameters such as root-mean-square deviation (RMSD) (Park et al, 2019), rootmean-square fluctuation (RMSF) (Narang et al, 2019), solvent accessible surface area (SASA) (Chingin & Barylyuk, 2018), and radius of gyration (Rg) (Lopes et al, 2019) were recorded for target structures. Simulation system components including water, ions, and spike protein were coupled to physiological temperature (37 C) and pressure (1 bar).…”