Ion Mobility-Collision Induced Unfolding Detects Stability Shifts within Hidden Protein Conformer Families

Abstract

Proteins commonly exist in complex mixtures, adopting a diverse ensemble of structural conformations. While some of these conformations are functionally important, others (e.g., preaggregative states), can adversely affect protein function. In monoclonal antibody (mAb)-based biotherapeutics such structural polydispersity is essential for native function but requires advanced biophysical and analytical techniques to assess and properly engineer optimal therapeutics. Methods such as native mass spectrometry (MS) and ion mobility-mass spectrometry (IM-MS) have become increasingly valuable for studying protein structure. Collision induced unfolding (CIU), an IM-MS assay, is frequently used to examine global higher-order structures of proteins. However, standard CIU assays are fundamentally limited by the IM resolution achieved for the precursor ion population, which may contain multiple conformers with similar collision cross sections. In this report, we use cyclic IM-MS (cIM-MS) to develop and conduct IM-selected CIU assays, which show dramatically enhanced capabilities for analyzing structurally polydisperse samples. To develop the IM-CIU approach, we investigate the model protein bovine serum albumin under both low and neutral pH conditions, demonstrating that IM-CIU can accurately identify domain III as the most perturbed region of the protein under these conditions. Additionally, we apply IM-CIU to analyze interleukin dimers, previously linked in therapeutic fusion protein constructs, and demonstrate that they exist in two distinct solution-based conformations. We conclude by outlining the fundamental principles of the IM-CIU method, highlighting its ability to resolve proteins with near-identical collision cross sections, and discussing its potential impact on the development of future pharmaceutical applications.