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.

Figure 1

A: Diagrams and cartoons depicting the CIU of proteins and common methods of analysis. As collision energy (eV) is increased, an isolated protein ion unfolds in the gas phase. B: CIU fingerprint with collision voltage on the x-axis, arrival time on y-axis, and intensity shown using a color scale. C: CIU comparison plot analysis depicting an apo and a doubly bound protein-ligand complex (red and green oval) with collision voltage on x-axis, arrival time on y-axis, and color scheme representing the differential intensities of the apo (red) and ligand bound (blue) states. D: A scaled deviation score analysis depicting a comparison of two different ligand bound states with CIU data acquired for the apo protein A score is computed that statistically assess fingerprint similarity at each voltage, enabling a narrow the window of collision voltage to be defined that maximizes dissimilarity between analytes, as shown by green shaded area for ligand and red shaded area for ligand.

Figure 2

A: A series of covalently linked poly-ubiquitin proteins (1–4 ubiquitins, gray spheres) is probed by CIU [37]**. Single domain ubiquitin results in a single CIU transition, from an initial native-like state (I) to a more extended state (II) upon collisional activation. Each additional domain added results in an additional CIU transition, indicating that the transitions are representative of the domain structure of the protein in solution. B: Bovine, human, and murine serum albumin proteins CIU fingerprints are compared. Despite high sequence homology and globally similar three-dimensional structures, CIU readily distinguishes each variant, demonstrating sensitivity towards subtle alterations in protein isoforms [13]**. C: Coefficient of variation (CV) across the bovine, human, and murine albumins represented in (B) for centroid voltage (blue), stability or horizontal length (red), and center drift time (green) for each feature. High CVs indicate significant differences between fingerprints. D: Comparison of type I (Dasantinib, left) and type II (Imatinib, right) inhibitors bound to Abl kinase. CIU distinguishes the binding location of inhibitors to the kinase, enabling a screening assay based on the region of maximal difference in the CIU fingerprint (far right) [17]. E: IgG subtypes 1–4 (left to right) are quantitatively distinguished by CIU [45]**. Each subtype exhibits different patterns of disulfide bonding in a broadly conserved overall structure, resulting in different CIU fingerprints.