Cytochrome c is Highly Compact upon Transfer into the Gas Phase from Strong Acids as Revealed by High-Pressure ESI Ion Mobility-Mass Spectrometry

Abstract

Abstract A substantial increase in the acidity of the protein solution (e.g., from pH 2 to pH 1) is known to induce the compaction of fully unfolded proteins, such as cytochrome c, into a non-native yet highly compact molten globule state, referred to as the A-state. In this study, the conformational landscape of the gaseous ions originating from the acid-induced A-state of cytochrome c, characterized by low charge states (6+ and 7+) with narrow charge-state distributions, is investigated by using high-pressure electrospray ionization ion mobility mass spectrometry (HP-ESI-IM-MS). HP ESI provides discharge-free, stable nanoESI-equivalent ionization for the A-state protein from highly conductive solutions. Collision cross section (CCS) analysis revealed that the dominant low-charge-state ions adopt highly compact conformations. Notably, the 6+ charge-state species obtained from trifluoroacetic acid at pH 1 exhibited an average CCS lower than that generated from native protein in pH-neutral ammonium acetate solutions. The HP-ESI-IM-MS results suggest that the acid-induced compaction in solution yields gas-phase structures that are as compact as or even more compact than those of the native state, offering new insights into the protein folding landscapes.