Characterizing Protein Solvent Accessible Surface Area in Solution by Dual Polarity Native Mass Spectrometry.

Native mass spectrometry (nMS) is rapidly emerging as a pivotal technique for exploring protein conformations and protein-ligand interactions. Pioneering research has demonstrated that the charge state distribution (CSD) of proteins in native mass spectra can be indicative of their solvent accessible surface area (SASA). Moreover, beyond SASA, it is postulated that the abundance of acidic and basic amino acids on the protein surface may also impact the CSD. Specifically, basic amino acids tend to acquire positive charges during electrospray ionization (ESI), whereas acidic amino acids are prone to adopting negative charges. Consequently, this study investigates the CSDs of globular proteins in both positive and negative ion modes to provide a comprehensive characterization of protein SASA. Experiments were conducted under both native ESI and native nano-ESI conditions. By harnessing the average charges observed across dual polarity nMS data, we achieved significantly enhanced log linear correlations between protein SASA and its CSDs. The coefficient of determination (R²) improved from 0.9866 to 0.9888 under ESI conditions and from 0.9677 to 0.9902 under nano-ESI conditions when compared to models utilizing only positive ion mode data. These findings suggest that the SASA of globular proteins can be effectively characterized through the CSDs derived from dual polarity nMS analysis.