pH Dependence of the Number of Discrete Conformers of Carbonic Anhydrase 2, as Evaluated from Collision Cross-Section Using Ion Mobility Coupled with Electrospray Ionization

Abstract

Ion mobility experiments coupled with electrospray ionization (ESI) were conducted to evaluate the folding states of bovine carbonic anhydrase 2 (CA2) under three different pH conditions. Collision cross-section (CCS) of the CA2 ions generated by ESI revealed the presence of six discrete conformers in the gas phase under the conditions employed in this study. The CCS of the most extended conformer was three times larger than that of the most compact one. The charge state distribution of the CA2 ions was indicative of three conformers being present. Although there was consistency in conformer assignment conducted by CCS and charge state distribution, the CCS measurement was shown to be more effective because the information obtained provided more detailed knowledge of the conformation of the protein.

Investigating direct current potentials that affect native protein conformation during trapped ion mobility spectrometry–mass spectrometry

“…The single bands observed at 43 V predominantly indicate a unimodal conformational distribution of BCA ions. The average CCS range from 2500 to 2625 Å 2 and slightly increase with protein charge, consistent with reported CCS values of native BCA 8,56,57 . This substantiates that post‐TIMS‐separation energetics do not alter the ion mobility profile.…”

Probing the Fundamentals of Native Liquid Extraction Surface Analysis Mass Spectrometry of Proteins: Can Proteins Refold during Extraction?

“…A bimodal distribution appears in the denatured mass spectra that suggest an expanded, flexible structure, potentially with multiple conformers. Nabuchi et al 65 noted that at pH 3.8 the mass spectrum of carbonic anhydrase shows a bimodal distribution, comprised of the apoprotein with a CSD of 11+ to 38+ and the holo (zincbound)-protein with a CSD of 10+ to 20+. In a follow-up study, 66 these researchers demonstrated that a drop in pH from 5.0 to 3.6 resulted in the loss of Zn 2+ and in significant unfolding of the N-terminus.…”