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.

The authors' abstract, as published at the source. Journal of the American Society for Mass Spectrometry, 2026 · DOI ↗