Collision Cross Sections for Native Proteomics: Challenges and Opportunities

Abstract

Recent advancements place a comprehensive catalog of protein structure, oligomeric state, sequence, and modification status tentatively within reach, thus providing an unprecedented roadmap to therapies for many human diseases. To achieve this goal, revolutionary technologies capable of bridging key gaps in our ability to simultaneously measure protein composition and structure must be developed. Much of the current progress in this area has been catalyzed by mass spectrometry (MS) tools, which have become an indispensable resource for interrogating the structural proteome. For example, methods associated with native proteomics seek to comprehensively capture and quantify the endogenous assembly states for all proteins within an organism. Such technologies have often been partnered with ion mobility (IM) separation, from which collision cross section (CCS) information can be rapidly extracted to provide protein size information. IM technologies are also being developed that utilize CCS values to enhance the confidence of protein identification workflows derived from liquid chromatography-IM-MS analyses of enzymatically produced peptide mixtures. Such parallel advancements in technology beg the question: can CCS values prove similarly useful for the identification of intact proteins and their complexes in native proteomics? In this perspective, I examine current evidence and technology trends to explore the promise and limitations of such CCS information for the comprehensive analysis of multiprotein complexes from cellular mixtures.

Performance evaluation of in-source ion activation hardware for collision-induced unfolding of proteins and protein complexes on a drift tube ion mobility-mass spectrometer

“…Importantly, the CIU fingerprints obtained for each protein are qualitatively similar across the different laboratories, in that all proteins sample similar intermediate CIU features, supporting the use of CIU fingerprints to support proteoform identification. 51…”

Symmetry of 4-Oxalocrotonate Tautomerase Trimers Influences Unfolding and Fragmentation in the Gas Phase

“…The intriguing formation and organization of salt bridges has motivated the exploration of new biophysical methods to further unveil the critical impact of these dynamic electrostatic interactions on the assembly and disassembly of proteins. Native mass spectrometry has emerged as a new technology for analysis of topologies and binding interactions of noncovalent macromolecular assemblies. − Transfer of compact folded structures from solution to the gas phase can be achieved using nanoelectrospray ionization (nESI), and preservation of native-like molecular architectures has been confirmed using ion mobility spectrometry (IMS). − IMS separates ions based on their size and shape and provides an extra dimension of conformational insight in addition to the mass and charge information provided by MS . Collision cross section (CCS) values can also be estimated from the decay of an ion population in Fourier transform mass analyzers, − termed transient decay analysis (TDA).…”

Performance Evaluation of In-source Ion Activation Hardware for Collision-Induced Unfolding of Proteins and Protein Complexes on a Drift Tube Ion Mobility-Mass Spectrometer

“…Importantly, the CIU fingerprints obtained for each protein are qualitatively similar across the different laboratories, in that all proteins sample similar intermediate CIU features, supporting the use of CIU fingerprints to support proteoform identification. 51 While these CIU fingerprints were found to be highly reproducible within each laboratory, there are interlaboratory differences observed in the CIU fingerprints we recorded, particularly with respect to various stable intermediate structural families, referred to as CIU "features" (F). For example, ubiquitin (+6) exhibits a clear population of intermediate conformers (~14 nm 2 CCS) which appear with different degrees of prominence across all three laboratory datasets (Figure 3A).…”