Thiol-ene Microfluidic Chip for Performing Hydrogen/Deuterium Exchange of Proteins at Subsecond Time Scales

Hydrogen/deuterium exchange monitored by mass spectrometry (HDX-MS) has become a routine approach for sensitive analysis of the dynamic structure and interactions of proteins. However, transient conformational changes and weak affinity interactions found in many biological systems typically only perturb fast-exchanging amides in proteins. Detection of HDX changes for such amides require shorter deuterium labeling times (subsecond) than can be performed reproducibly by manual sample handling. Here, we describe the development and validation of a microfluidic chip capable of rapid on-chip protein labeling and reaction quenching. The fastHDX thiol-ene microchip is fabricated entirely using thiol-ene photochemistry. The chip has a three-channel design for introduction of protein sample, deuterated buffer, and quench buffer. Thiol-ene based monolith plugs (i.e., polymerized thiol-ene emulsions) situated within microchannels are generated in situ using a 3D-printed photolithography mask. We show that efficient on-chip mixing can be achieved at channel junctions by spatially confined in-channel monolith mixers. Using human hemoglobin (Hb), we demonstrate the ability of the chip to perform highly reproducible HDX in the 0.14-1.1 s time frame. The HDX of Hb at 0.14-1.1 s, resolved to peptide segments, correlates closely with structural features of the crystal structure of the Hb tetramer, with helices exhibiting no or minor HDX and loops undergoing pronounced HDX even at subsecond time scales. On-chip HDX of Hb at time points ranging from 0.14-1.1 s demonstrates the ability to distinguish fast exchanging amides and thus provides enhanced detection of transient structure and interactions in dynamic or exposed regions of proteins in solution.