Quantitative Hydrogen/Deuterium Exchange Mass Spectrometry

Area of Science:

• Biophysical Chemistry
• Analytical Chemistry
• Structural Biology

Background:

• Hydrogen/deuterium exchange-mass spectrometry (HDX-MS) is a powerful biophysical technique.
• HDX-MS data interpretation often remains qualitative, limiting quantitative insights.
• There is a need for robust methodologies to derive quantitative data from HDX-MS.

Purpose of the Study:

• To provide a comprehensive guide for generating, analyzing, and interpreting HDX-MS data for quantitative arguments.
• To address limitations in current HDX-MS methodologies for obtaining precise biophysical data.
• To improve the reliability and accuracy of HDX-MS perturbation studies.

Main Methods:

• Optimizing sequence coverage and resolution during the feasibility stage.
• Enhancing time window coverage and resolution during the hydrogen/deuterium exchange (HDX) stage.
• Utilizing centroid values and isotope envelopes for quantitative data extraction from medium-resolution HDX-MS data.

Main Results:

• Strategies for improving sequence and time window coverage to minimize false negatives.
• Methods for increasing sequence resolution to reduce false positives.
• Techniques for extracting quantitative biophysical values from deuterium buildup curves and isotope envelopes.

Conclusions:

• Careful examination of potential errors, including false negatives from coverage gaps and false positives from resolution or allosteric effects, is crucial.
• Implementing optimized data generation and analysis protocols leads to more accurate quantitative arguments in HDX-MS.
• This work facilitates the transition of HDX-MS from a qualitative to a more quantitative biophysical tool.