Characterizing the Structures and Folding of Free Proteins Using 2-D Gas-Phase Separations: Observation of Multiple Unfolded Conformers
Understanding the 3-D structure and dynamics of proteins and other biological macromolecules in various environments is among the central challenges of chemistry. Electrospray ionization (ESI) can transfer ions from solution to gas phase with only limited structural distortion, allowing their profiling using mass spectrometry and other gas phase approaches. Ion mobility spectrometry (IMS) can be used to separate and characterize macroion conformations with high sensitivity and speed. However, IMS separation power has proven insufficient for full resolution of major structural variants of protein ions and elucidation of their interconversion dynamics. Here we report characterization of macromolecular conformations using field asymmetric waveform IMS (FAIMS) coupled to conventional IMS in conjunction with mass spectrometry. The controlled activation of ions in the electrodynamic funnel trap between FAIMS and IMS stages enables investigating the structural evolution of particular isomeric precursors as a function of the extent and duration of activation that can be varied over a large range. These new capabilities are demonstrated for bovine ubiquitin, a common model for study of structure and folding of gas-phase proteins. For nearly all charge states, two-dimensional FAIMS/IMS separations distinguish many more conformations than either FAIMS or IMS alone, including some species with very low abundance. The unfolding of specific ubiquitin conformers has been studied employing ion heating in the FAIMS/IMS interface.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 883673
- Report Number(s):
- PNNL-SA-46452; 400412000; TRN: US200614%%887
- Journal Information:
- Analytical Chemistry, 78(10):3304-3315, Vol. 78, Issue 10
- Country of Publication:
- United States
- Language:
- English
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