Microfluidic Platform for Time-Resolved Characterization of Protein Higher-Order Structures and Dynamics Using...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Structural Biology

Background:

• Protein higher-order structures and dynamics are crucial for biological function.
• Top-down mass spectrometry (MS) provides intact protein and fragment-level structural insights.
• Native top-down MS and hydrogen/deuterium exchange (HDX) MS offer advanced capabilities for analyzing protein complexes and conformations.

Purpose of the Study:

• To design and demonstrate a microfluidic chip tailored for top-down MS analysis of protein higher-order structures and dynamics.
• To enable time-resolved native MS and HDX MS workflows with enhanced control and efficiency.
• To overcome limitations of existing microchip-MS devices primarily designed for bottom-up approaches.

Main Methods:

• Development of a microchip for top-down MS with efficient ionization of intact protein complexes.
• Implementation of flexible manipulation of multiple reactant flows and precise control of reaction times.
• Application of the microchip to time-resolved native MS and HDX MS analyses.

Main Results:

• Demonstrated successful application of the microchip for analyzing monoclonal antibodies and antibody-antigen complexes.
• Showcased the ability to characterize coexisting protein conformers with high spatial resolution.
• Validated the microchip's performance across a broad range of submicroliter per minute flow rates.

Conclusions:

• The developed microchip strategy is effective for top-down MS analysis of protein higher-order structures and dynamics.
• This approach facilitates elaborate structural analysis of biomacromolecules.
• The microchip design offers a promising platform for future method development in MS-based structural biology.