Gas-phase processes and measurements of macromolecular properties in solution: On the possibility of false positive and false negative signals of protein unfolding

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Abstract Electrospray ionization mass spectrometry is increasingly applied to study protein behavior in solution, including characterization of their higher order structure, conformational dynamics and interactions with small ligands and other biopolymers. However, actual measurements of fundamental ionic parameters (mass and charge) take place in vacuum, and an array of gas phase processes occurring prior to protein ion detection and characterization may certainly affect them. While most previous studies were concerned primarily with the effect of gas phase processes on mass measurement (e.g., integrity of macromolecular complexes in the absence of solvent, non-specific interactions, etc.), the focus of our attention is the ionic charge. Charge state distributions of protein ions in ESI MS are often used to characterize large-scale dynamic processes in solution (such as protein unfolding). Formation of metastable protein aggregates either in the bulk of solution or in the electrosprayed droplets, their consequent transfer to the gas phase and asymmetric dissociation may give rise to a population of highly charged ions. Presence of such ions in ESI mass spectra usually indicates loss of native structure in solution. Therefore, studies of large-scale conformational dynamics in solution by monitoring protein ion charge state distributions should be carried out at low protein concentrations in order to minimize the occurrence of false positive signals of protein unfolding. The opposite phenomenon, absence of highly protonated ionic species in ESI spectra of unfolded proteins, does not occur even in the case of highly acidic proteins lacking a sufficient number of basic residues.