Nanoscaled, Fully Automated Hydrogen/Deuterium Exchange for Analysis of Macromolecular Assemblies Reaching the MDa Scale

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) has become an indispensable tool for structural biology, yet conventional microflow configurations limit its application to readily expressible proteins available in microgram quantities. Here, we present nanoscaled HDX, a nanoflow implementation on a commercial automated HDX platform that overcomes these limitations. We demonstrate that rapid, online microflow digestion and trapping can be effectively coupled with nanoflow separation at low temperatures to achieve unprecedented sensitivity. By integrating narrower bore tubing, optimized valve configurations, and appropriate columns, we achieved a >20-fold reduction in gradient delay volume while enhancing sensitivity by over 100-fold, maintaining equivalent performance while using just 10 ng versus 1 μg of peptide mix per injection. We demonstrate exceptional system robustness with chromatographic reproducibility below 1.5% CV for peptides, retention time shifts averaging 0.035 min, deuterium (D)-uptake measurements with SDs of 0.1 Da and improved D-retention compared to conventional microflow HDX. The drastic reduction in the amounts of protein required enabled characterization of challenging macromolecular complexes previously inaccessible to conventional HDX-MS. Using 250 fmol of the 320 kDa polycomb repressive complex 2 per injection, we achieved sequence coverage exceeding 86%, while the 0.9 MDa RNA editing catalytic complex 2, generated following one-step purification, yielded 77% coverage with 600 fmol of RNA editing catalytic complex 2 per injection. The nanoscaled HDX configuration reduces sample requirements to the sub-pmol range per injection without compromising performance relative to conventional HDX-MS, enabling the analysis of previously intractable protein systems with limited sample availability or those available only from rapid, low-yield purification protocols. Our straightforward implementation on a commercial platform eliminates the need for extensive method development, making this enhancement readily accessible and scalable to the broader structural biology community.