Practical Fragments

Best Practices for Applying HDX-MS to FBLD

A recent open‑access study demonstrates that hydrogen‑deuterium exchange mass spectrometry (HDX‑MS) can reliably map binding sites of extremely weak fragment hits (up to 7 mM KD) against Cyclophilin D. By optimizing protein concentration at 10 µM and testing fragments at 2.5‑10 mM, the authors observed measurable deuterium protection even with 5‑18% occupancy, confirmed through statistical validation. The method correctly identified aniline and pyrazolo pocket binders, uncovered a pH‑dependent proline‑pocket interaction, and reproduced known binding modes for a FAK fragment. The work provides a practical roadmap for applying HDX‑MS in fragment‑based lead discovery.