Examining Instrumental Factors Influencing the Performance of Data-Independent Acquisition Methods in Hydrogen/Deuterium Exchange Mass Spectrometry

Hydrogen/deuterium exchange mass spectrometry (HX-MS) is a method used to study solution-phase protein structure and dynamics. Despite its many applications, HX-MS is limited in throughput because manual data analysis is still the norm. We previously developed HX-MS² technology to add a second dimension of deuteration data and promote automated data processing. Data-independent acquisition (DIA) techniques enable this approach, but we require optimized methods for best performance. Using an Orbitrap Eclipse for illustration, we show that ion optics and collision energy settings typical of a proteomics DIA experiment generate maximal peptide retrieval from the DIA library. As few as three MS² sequence ions are sufficient to generate a deuteration measurement with a precision that exceeds what is possible in traditional HX-MS. DIA window sizes are based on the chromatographic resolution of the method. An inter-scan window offset method is the recommended default configuration for most HX-DIA applications butan intra-scan overlap method can be tuned for highest performance and is recommended when maximum peptide retrieval is desired. We demonstrate the robustness of one HX-MS² configuration (consisting of Trajan HDX automation technology, an Orbitrap Eclipse mass spectrometer and AutoHX software) on an extensive time-course analysis of phosphorylase B and an epitope analysis of single-domain antibodies (V_HHs, nanobodies) specific to the receptor binding domain of SARS-CoV-2 spike protein.