Analysis of Temperature-Dependent H/D Exchange Mass Spectrometry Experiments

Area of Science:

• Biochemistry
• Structural Biology
• Mass Spectrometry

Background:

• Hydrogen/Deuterium (H/D) exchange mass spectrometry (HDX-MS) is crucial for studying protein structure and dynamics.
• Traditional HDX-MS uses constant temperature, limiting its ability to assess temperature-dependent protein stability, especially for therapeutics.
• Interpreting temperature-dependent HDX-MS data is challenging due to the difficulty in separating chemical labeling rates from protein dynamics.

Purpose of the Study:

• To develop a strategy for dissecting temperature-dependent HDX-MS profiles.
• To separate contributions from the chemical labeling step (k_ch(T)) and protein dynamics (local and global).
• To enable analysis of protein dynamics across a wide temperature range.

Main Methods:

• Applied a novel analysis to temperature-dependent HDX-MS data from myoglobin.
• Dissected HDX-MS profiles into temperature-dependent chemical labeling rates and protein dynamics.
• Analyzed contributions of local and global protein fluctuations to deuterium uptake.

Main Results:

• Experimental HDX-MS profiles showed initially shallow slopes, deviating from expected temperature dependence.
• A sharp increase in deuterium uptake was observed just below the melting temperature (Tm).
• Local dynamics dominated at lower temperatures, while global dynamics became significant near Tm; about half of sites showed canonical thermodynamic parameters, while others exhibited negative ΔH and ΔS.

Conclusions:

• The study provides a method to analyze temperature-dependent HDX-MS data, separating chemical and dynamic contributions.
• This approach allows for the characterization of protein dynamics and stability across a broad temperature spectrum.
• Enables future high-throughput screening applications of HDX-MS for temperature-dependent protein analysis.