Enabling simultaneous photoluminescence spectroscopy and X-ray footprinting mass spectrometry to study protein...

X-ray footprinting mass spectrometry (XFMS) is a structural biology method that uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation in solution. However, while XFMS alone provides important structural information on biomolecules, as we move into the era of the interactome, hybrid methods are becoming increasingly necessary to gain a comprehensive understanding of protein complexes and interactions. Toward this end, we report the development of the first synergetic application of inline and real-time fluorescent spectroscopy at the Advanced Light Source's XFMS facility to study local protein interactions and global conformational changes simultaneously. To facilitate general use, we designed a flexible and optimum system for producing high-quality spectroscopy-XFMS hybrid data, with rapid interchangeable liquid jet or capillary sample delivery for multimodal inline spectroscopy, and several choices for optofluidic environments. To validate the hybrid system, we used the covalently interacting SpyCatcher-SpyTag split protein system. We show that our hybrid system can be used to detect the interaction of SpyTag and SpyCatcher via fluorescence resonance energy transfer (FRET), while elucidating key structural features throughout the complex at the residue level via XFMS. Our results highlight the usefulness of hybrid method in providing binding and structural details to precisely engineer protein interactions.