Cyclic Ion Mobility–Collision Activation Experiments Elucidate Protein Behavior in the Gas Phase

Abstract

Ion mobility coupled to mass spectrometry (IM-MS) is widely used to study protein dynamics and structure in the gas phase. Increasing the energy with which the protein ions are introduced to the IM cell can induce them to unfold, providing information on the comparative energetics of unfolding between different proteoforms. Recently, a high-resolution cyclic IM-mass spectrometer (cIM-MS) was introduced, allowing multiple, consecutive tandem IM experiments (IM n ) to be carried out. We describe a tandem IM technique for defining detailed protein unfolding pathways and the dynamics of disordered proteins. The method involves multiple rounds of IM separation and collision activation (CA): IM-CA-IM and CA-IM-CA-IM. Here, we explore its application to studies of a model protein, cytochrome C, and dimeric human islet amyloid polypeptide (hIAPP), a cytotoxic and amyloidogenic peptide involved in type II diabetes. In agreement with prior work using single stage IM-MS, several unfolding events are observed for cytochrome C. IM n -MS experiments also show evidence of interconversion between compact and extended structures. IM n -MS data for hIAPP shows interconversion prior to dissociation, suggesting that the certain conformations have low energy barriers between them and transition between compact and extended forms.

Collision-Induced Unfolding of Native-like Protein Ions Within a Trapped Ion Mobility Spectrometry Device

“…The unfolding pattern observed for the 7+ ion resembles previously acquired data collected on a cyclic IM device but the improved resolution of the TIMS enables the differentiation of several conformers not identified in this previous work (Figure 3C). 35,36 The 8+ ion undergoes extensive unfolding over a narrow range of energies (Figure 3B). While this rapid conversion to a single dominate extended structure has been observed before, 37 TIMS CIU uncovers two minor structural features at approximately 2300 and 2340 Å 2 that as far as we are aware, have not been previously resolved (Figure 3B).…”

Recent technological developments for native mass spectrometry

“…28 CIU fingerprints showed distinct differences for gain and loss of function mutations of the voltage sensor domain, illustrating the ability of CIU to discriminate between various functional forms due to their effects on the overall gas-phase stability of the protein ions. Newly commercially available tandem TIMS 31 and cyclic TWIMS 32 devices are allowing many users to not only perform traditional pre-mobility CIU experiments, but also to select specific conformer families in CCS space, activate them (or not) and further resolve these activated and/or selected conformers, giving high structural specificity to gas-phase protein characterization. For example, Figure 1 shows CIU fingerprints that detail the evolution of nativelike and collisionally activated cytochrome c 7 + ion structures.…”

“…For example, Figure 1 shows CIU fingerprints that detail the evolution of nativelike and collisionally activated cytochrome c 7 + ion structures. 32 Each of the observed subpopulations was isolated by mobility and activated at various collision energies, allowing for the gas-phase unfolding of each subpopulation to be monitored. Surface induced dissociation (SID), where ions are accelerated into a surface, a much more massive target than a gas molecule, extensively fragments protein complexes before structural rearrangement can occur.…”

“…The * denotes the low intensity species mobility selected in plot (D). Reproduced with permission from ref 32 https://pubs.acs.org/doi/10.1021/jasms.1c00018. Copyright 2021 American Chemical Society.…”

Exploiting Self-Association to Evaluate Enantiomeric Composition by Cyclic Ion Mobility–Mass Spectrometry

“… 12 It has shown early promise in the characterization of isomeric species, including oligosaccharides, 34 − 39 nucleosides, 40 peptides, 41 − 43 fuels, 44 − 46 and native proteins. 47 , 48 In particular, it is unique in its ability to perform many rounds of mobility separation punctuated by fragmentation steps, termed IMS n . 12 , 37 Despite the ultrahigh resolution of cyclic TWIMS and any other ion mobility technique, it does not enable separation of enantiomers due to the requirement of a chiral separation environment.…”