Collision induced unfolding of isolated proteins in the gas phase: past, present, and future

Abstract

Rapidly characterizing the three-dimensional structures of proteins and the multimeric machines they form remains one of the great challenges facing modern biological and medical sciences. Ion mobility-mass spectrometry based techniques are playing an expanding role in characterizing these functional complexes, especially in drug discovery and development workflows. Despite this expansion, ion mobility-mass spectrometry faces many challenges, especially in the context of detecting small differences in protein tertiary structure that bear functional consequences. Collision induced unfolding is an ion mobility-mass spectrometry method that enables the rapid differentiation of subtly-different protein isoforms based on their unfolding patterns and stabilities. In this review, we summarize the modern implementation of such gas-phase unfolding experiments and provide an overview of recent developments in both methods and applications.

Quantitative collision‐induced unfolding differentiates model antibody–drug conjugates

“…The combination of ion mobility (IM) and MS has proven to be a useful tool for the characterization of mAbs and ADCs, by separating such proteins according to their size and recording their ion‐neutral collision cross sections (CCSs) as a means of HOS analysis . In an effort to assess antibody structures in greater detail, collision induced unfolding (CIU) experiments can be performed . This technology involves the collisional activation of protein ions prior to IM‐MS separation in order to initiate protein unfolding events in the gas‐phase.…”

“…15,[26][27][28] In an effort to assess antibody structures in greater detail, collision induced unfolding (CIU) experiments can be performed. 29 This technology involves the collisional activation of protein ions prior to IM-MS separation in order to initiate protein unfolding events in the gas-phase. CIU is capable of differentiating IgG subclasses, detecting minor alterations in mAb glycoforms, assessing stability shifts associated with site-specific ADCs, and evaluating the comparability of biosimilars.…”

Collision-Induced Unfolding Reveals Unique Fingerprints for Remote Protein Interaction Sites in the KIX Regulation Domain

“…Additionally, collision induced dissociation (CID) can be used to measure the ejection of ligands from protein:ligand complexes [25]. CIU has been used to distinguish between modes of ATP-competitive kinase inhibitors [26], in biosimilar analysis [27, 28], to assess biotherapeutics [29], and to probe the binding of specific lipids to membrane protein complexes [30-34]. In this report, we develop a method using CIU to differentiate between KIX:peptide(MLL, E2A, pKID, and c-Myb) complexes.…”

Phosphorylation of HspB1 regulates its mechanosensitive molecular chaperone interaction with native filamin C

“…As a proxy for mechanical stress, we performed gas-phase experiments in which protein ions are activated by collisions with inert gas, leading to conformational perturbations and Coulombic-repulsion-driven chain extension (64–66). Providing an analogous endpoint to mechanical unfolding, an advantage of this approach is that, by capitalising on the sensitivity and capability of ion selection in the mass spectrometer, even low-affinity complexes can be interrogated individually (67).…”