The automation of nanoHX for sub-pmol amounts of protein

Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a powerful technique for probing protein structure and dynamics; however, its broader application in pharmaceutical research has been constrained by limited sensitivity and high sample requirements. Our earlier nanospray HDX-MS system improved sensitivity but lacked automation, robustness, and online digestion. Here, I present a robust NanoHD source compatible with Thermo Scientific EasySpray mass spectrometers. The prototype enables a substantial reduction in LC separation flow rates to the nL/min regime, compared with conventional HDX-MS workflows operating at µL/min. This nanoflow LC configuration enhances analytical sensitivity. To support digestion strategies under these conditions, a miniaturized immobilized enzyme reactor (IMER) was developed for online digestion, and in-solution digestion was also evaluated to optimize digestion performance under NanoHD conditions. The results demonstrate that both IMER-based and in-solution digestion are compatible with nanoflow HDX-MS. The NanoHD source was subsequently integrated with an autosampler and the IMER, enabling fully automated operation with improved sensitivity, reduced sample consumption, and reliable routine use. Using this platform, we achieved near-complete sequence coverage with only 250 fmol of phosphorylase B and Eg5, and 0.5 pmol of membrane proteins such as dimeric BlaR1 and the TagH/TagG complex—approximately 100-fold less protein than required for conventional HDX-MS workflows. Low back-exchange (~25%) at these ultralow flow rates demonstrates the effectiveness of the system design and its suitability for routine and high-throughput applications. Fully automated measurements were further demonstrated by investigating the interaction between FEN1, a cancer-relevant target, and a small-molecule inhibitor. The NanoHD platform produced HDX-MS results for 0.5 pmol of FEN1 that were comparable to those obtained using approximately 80 pmol in a conventional workflow, representing a ~160-fold improvement in sensitivity. Overall, this automated nanoflow HDX-MS workflow substantially reduces sample requirements while maintaining high analytical performance, extending the applicability of HDX MS to scarce or complex proteins in biopharmaceutical and structural biology research.