Two-dimensional ion mobility analyses of proteins and peptides

Area of Science:

• Proteomics and structural biology
• Analytical chemistry
• Biophysical characterization

Background:

• Ion mobility spectrometry (IMS) is crucial for mass spectrometry of proteins and peptides.
• IMS offers high-speed separations, enhancing proteomic analysis throughput and peak capacity.
• Existing IMS methods include drift-tube IMS (absolute mobility) and FAIMS (differential mobility).

Purpose of the Study:

• To develop and demonstrate a novel 2D separation technique combining FAIMS and IMS.
• To improve the characterization of protein structure and dynamics.
• To advance proteomic analysis by increasing separation power and dynamic range.

Main Methods:

• Integration of FAIMS and drift-tube IMS into a 2D separation platform coupled with time-of-flight mass spectrometry.
• Utilization of electrodynamic ion funnel interfaces, including "hourglass" funnels, for efficient ion transfer and accumulation.
• Controlled ion heating in a funnel trap between FAIMS and IMS stages for thermodynamic and kinetic studies.

Main Results:

• Achieved a peak capacity of approximately 500 for peptide separations, comparable to leading liquid chromatography systems.
• Enabled the distinction of more protein conformers than either IMS technique alone.
• Extended the dynamic range of detection by an order of magnitude compared to 1D IMS.
• Demonstrated the ability to follow the evolution of selected isomers, providing insights into protein folding pathways.

Conclusions:

• The combined FAIMS/IMS 2D separation offers significant advantages for both high-throughput proteomics and detailed structural biology.
• This integrated approach provides enhanced capabilities for distinguishing protein conformers and analyzing folding dynamics.
• The developed methodology opens new avenues for mapping protein folding pathways and energy landscapes.