Collisional and Coulombic Unfolding of Gas‐Phase Proteins: High Correlation to Their Domain Structures in Solution

Abstract

The three-dimensional structures adopted by proteins are predicated by their many biological functions. Mass spectrometry has played a rapidly expanding role in protein structure discovery, enabling the generation of models for both proteins and their higher-order assemblies. While important coursed-grained insights have been generated, relatively few examples exist where mass spectrometry has been successfully applied to the characterization of protein tertiary structure. Here, we demonstrate that gas-phase unfolding can be used to determine the number of autonomously folded domains within monomeric proteins. Our ion mobility-mass spectrometry data highlight a strong, positive correlation between the number of protein unfolding transitions observed in the gas phase and the number of known domains within a group of sixteen proteins ranging from 8-78 kDa. This correlation and its potential uses for structural biology is discussed.

Coming to Grips with Ambiguity: Ion Mobility-Mass Spectrometry for Protein Quaternary Structure Assignment

“…Our CG error analysis (Figure 2) clearly motivates the development of domain-level IM-MS models of protein quaternary structure, and a move away from CG at the intact subunit level. The development of IM-MS tools for the generation of such information on protein tertiary structure, such as collision induced unfolding (CIU),[58,59] as well as efforts to integrate IM-MS data with other sources of experimental data sensitive to local protein structure[60,61,51] and computational domain assignment algorithms[62] will, therefore, become increasingly important in future IM-MS protein topology modeling efforts. Similarly, our analysis of ambiguity in IM-MS models of protein quaternary structure strongly points to the need for improved methodologies capable of detecting protein complex connectivity and symmetry.…”

Towards the analysis of high molecular weight proteins and protein complexes using TIMS-MS

“…[41] This description holds true for most contemporary IMS analyzers (e.g., periodic focusing DC ion guide [42–44], segmented quadrupole drift cell [45], multistage IMS [46–48], traveling wave ion guide (TWIMS) [49, 50], and SLIM devices [51]), and a common pursuit has been to increase IMS resolving power and ion transmission. [52–60] Many different forms of IMS have been used in the analysis of biological molecules for the analysis of isotopomers[61], proteins[62, 63], protein complexes[64–71], folding pathways[72–74], unstructured/intrinsically disordered proteins[75–79], as well as collisionally activated states of peptides and proteins[69, 80–87]. It should be noted that, in the case of structural biology, gas-phase studies take advantage of the desolvation process to effectively reduce sample complexity, permitting molecular characterization in the absence of bulk solvent.…”

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

“…Specifically, CIU fingerprints recorded for 7+ KIX ions exhibited fewer structural transitions upon activation, and 9+ ions exhibited larger CCS values in comparison to lower charge states prior to CIU (data not shown). Similar criteria have been used previously to select charge states for CIU assay development [26, 27, 42]. …”