An Ultra-High Throughput Hydrogen–Deuterium Exchange Workflow Using Acoustic Ejection Mass Spectrometry for Studying Peptide Solution Structural Conformation

Peptides have become an important modality in the pharmaceutical industry, as they demonstrate heightened potency and target specificity, a property influenced by their conformational structures in solutions. In early drug discovery, structure-based optimization of peptide solution conformation in iterative make-test-design cycles to access peptide drug leads involves large-scale screening. To date, workflows for studying peptide solution conformation reported in the literature are throughput- and resource-limited and do not meet the analytical needs in early discovery space. In this view,context, it is imperative to develop a methodology that exhibits a high analytical throughput, does not involve additional sample preparation steps, and can handle screening of large numbers of samples for solution conformation study. These important analytical requirements in an early discovery pipeline were addressed in this newly developed workflow, where hydrogen-deuterium exchange mass spectrometry (HDX-MS) was employed to gain insights into the relative solution structural conformation of peptides with ultra-high throughput (uHT) acoustic ejection mass spectrometry (AEMS). The described method exhibits a sampling rate of ∼3 s/sample with very low sample and solvent consumption. The format of this workflow provides the ease and convenience of investigating peptide HDX kinetic profiles, with negligible unwanted back-exchange. The capability of the workflow to discriminate conformational changes was established using myoglobin and apo-myoglobin as protein model systems. Using model peptides, we have further demonstrated the capability of the workflow to differentiate solution structural conformation based on their HDX profiles. This work is an example of a HT HDX-based solution structural conformation workflow that does not require additional sample preparation steps, using uHT AEMS with analytical characteristics suitable for a large sample size screening as part of the down-selection process in the early discovery stage.