Mass Spectrometry-Based Protein Footprinting for Protein Structure Characterization

Area of Science:

• Biochemistry and Structural Biology
• Analytical Chemistry
• Mass Spectrometry

Background:

• Protein higher-order structure (HOS) is crucial for biological function, with three-dimensional structures encoding protein machinery mechanisms.
• Mass spectrometry (MS)-based protein footprinting is an emerging technique for characterizing protein HOS by mapping solvent-accessible regions and hydrogen bonding.
• This method provides insights into protein dynamics, conformational changes, and interactions, especially when applied differentially to study responses to perturbations.

Purpose of the Study:

• To provide an account of the development and applications of MS-based protein footprinting techniques.
• To highlight the utility of fast and slow footprinting methods, including fast photochemical oxidation of proteins (FPOP) and hydrogen-deuterium exchange (HDX).
• To showcase the application of protein footprinting in studying challenging systems like biotherapeutics, metal-bound proteins, amyloid proteins, and integral membrane proteins (IMPs).

Main Methods:

• Development of fast footprinting methods using reactive reagents (e.g., FPOP) with reaction rates faster than protein folding/unfolding.
• Description of slow footprinting and hydrogen-deuterium exchange (HDX) for specific side-chain labeling and structural context.
• Application of complementary footprinting approaches to address limitations of high-resolution techniques for complex biological systems.

Main Results:

• Fast footprinting methods capture structural changes without biasing information due to rapid reaction kinetics.
• Slow footprinting and HDX provide valid structural information, often without requiring specialized apparatus.
• Successful application of footprinting to elucidate structures of biotherapeutics, metal-bound proteins, amyloid oligomers, and integral membrane proteins (IMPs).

Conclusions:

• MS-based protein footprinting is a versatile and powerful tool for characterizing protein HOS, dynamics, and interactions.
• Complementary fast and slow footprinting approaches, including HDX, are essential for comprehensive structural analysis.
• Protein footprinting offers unique advantages for studying complex and challenging biological systems where other methods fall short.