Effects of Charge State on the Structures of Serum Albumin Ions in the Gas Phase: Insights from Cation-to-Anion Proton-Transfer Reactions, Ion Mobility, and Mass Spectrometry.

Abstract

Understanding the structures of proteins in the gas phase is essential for using gas-phase measurements to infer the properties of proteins in solution. Using serum albumin as a model, this study aims to expand our understanding of this relationship for a larger (66 kDa), multidomain protein that contains 17 internal disulfide bonds. Gas-phase ions were generated from five solutions that preserve varying extents of the native structure. Ion mobility (IM) mass spectrometry, cation-to-anion proton-transfer-reactions (CAPTR), and energy-dependent IM were used to probe the relationship between structure, charge, and solution. Ions generated from increasingly disruptive conditions exhibited higher charge states and larger collision cross-section values. The collision cross-sections of all CAPTR products depend on the original solution and to varying extents the charge state of the product and the precursor. For example, the collision cross-sections of CAPTR products from denaturing conditions are all significantly larger than those of the original native-like ions. Results from energy-dependent experiments show that the structures of the original ions from electrospray ionization and their CAPTR products are a consequence of kinetic trapping and depend on higher-order structure and disulfide bonding in solution. This study builds on our understanding of the relationship between solution condition, disulfide bonding, collision cross-section, and charge for a larger, multidomain protein, which may be applicable for future characterization of biotherapeutics that share these structural features.