Site-Specific Hydrogen Deuterium Exchange Difference Mass Spectrometry Measurements for Ligand Binding

Conventional bottom-up HDX-MS experiments are highly suitable for use in drug development; however, a major limitation of this approach is that it generally provides only peptide-level structural resolution. Site specific (i.e., single amino acid-resolved) HDX-MS measurements have been achieved using ECD/ETD, but the low efficiency of these fragmentation techniques, combined with poor ion transmission associated with ‘detuning’ the instrument to fully prevent deuterium scrambling, results in sensitivity losses that make ligand binding measurements impractical in a ‘real-world’ (e.g., drug development) context. Here we apply a recently developed method for zero scrambling, high efficiency ECD in the challenging context of ligand binding differential HDX experiments, demonstrating a wealth of additional information that can be acquired when HDX-MS analyses are conducted at the amino acid level.