Analyzing HDX experiments in BioPharma Finder
Hydrogen deuterium exchange (HDX) mass spectrometry (MS) experiments take advantage of the labile nature of amide protons present on protein backbone, which can exchange with deuterium when dissolved in a deuterated buffer solution. HDX-MS can be used to measure the rate of hydrogen-deuterium exchange and obtain information on structure, protein-protein or protein-ligand interaction sites, allosteric effects, intrinsic disorder, and conformational changes induced by post-translational modifications (PTMs).
A typical HDX experiment involves comparing the conformation of a protein under different deuteration conditions. In the BioPharma Finder application, you can compare up to 26 different conditions, specifying each conformation with a unique letter name, A to Z.
Protection factor χ2 modeling
The protection factor (P) of a given proton refers to the ratio of its intrinsic exchange rate (kint) in a fully solvent-exposed state (typically obtained from a model system) to its exchange rate in some variable intermediate state of the protein (kobs).
That is, P = kint/kobs.
In an HDX Peptide Mapping Analysis experiment, the application generates a protection factor for each amide proton and displays the protection factor plot on the Mapping > Hydrogen Deuterium Exchange page after processing.
To avoid over-interpretation of the data when generating protection factors, the application minimizes the χ2 difference between the experimental and simulated data.
Back-exchange χ2 modeling
Back-exchange refers to the loss of deuterium labeling during sample preparation, separation, analysis. This phenomenon can lead to inaccuracy in HDX-MS experiments.
In an HDX Peptide Mapping Analysis experiment, the application calculates the effective back-exchange percentages at the proteolytic digestion and LC separation time points and then accounts for the back-exchange when generating protection factors.
Similar to the χ2 protection factor modeling, the application minimizes the χ2 difference between the experimental and simulated data for the fully deuterated control samples when calculating back-exchange.