Rigorous Analysis of Multimodal HDX-MS Spectra.

Area of Science:

• Biochemistry and Biophysics
• Analytical Chemistry
• Structural Biology

Background:

• Hydrogen/deuterium exchange coupled to mass spectrometry (HDX-MS) is a powerful technique for studying protein dynamics.
• HDX-MS can detect multiple protein conformational states, often observed as multimodal isotopic envelopes in mass spectra.
• Established statistical methods for accurately analyzing these multimodal spectra are lacking.

Purpose of the Study:

• To develop and validate a statistical approach for the accurate detection and deconvolution of multimodal isotopic distributions in HDX-MS data.
• To provide a robust computational tool for interpreting complex HDX-MS spectra arising from mixed protein populations or complex kinetic events.

Main Methods:

• An unrestrained binomial distribution fitting approach was developed to analyze isotopic distributions.
• Statistical tests were implemented to assess the significance of detected subpopulations.
• The algorithms were integrated into the updated HX-Express software and validated using known peptide mixtures.

Main Results:

• The developed approach accurately detects and deconvolutes multimodal isotopic envelopes in HDX-MS spectra.
• Validation using known peptide mixtures demonstrated the reliability of the method.
• The updated HX-Express software provides a user-friendly tool for analyzing complex HDX-MS data.

Conclusions:

• This study presents a statistically sound method for analyzing multimodal HDX-MS spectra.
• The enhanced HX-Express software facilitates the interpretation of protein conformational heterogeneity and complex exchange kinetics.
• This readily accessible tool will advance the application of HDX-MS in structural biology and biophysics.