PROJECT SUMMARY My laboratory innovates mass spectrometry (MS) technology to accelerate the pace, depth, and accuracy of proteome analysis and applies these technologies to globally access proteome structure, function, and regulation. Signature achievements include the development of electron-transfer dissociation (ETD), the GC- Orbitrap mass spectrometer, novel protein quantification technologies, and the targeted technique parallel reaction monitoring – to name a few. Having established our reputation in proteomics instrumentation, our mission came to include the investigation of the metabolome and lipidome. The rationale is simple: fully understanding a biological system requires knowing the interplay between molecular classes. We have demonstrated the capability of these technologies to propel numerous projects in the fields of metabolism, developmental, and systems biology. Building on my program’s expertise in the analysis of disassembled molecular machinery (i.e., shotgun proteomics), we have turned our attention to the machine as a whole, i.e., protein structure. By combining technologies from MS and cryogenic electron microscopy (cryo-EM) our aim is to maximize the information garnered from a single sample to provide insight into protein structure with unprecedented speed and simplicity. That is, we compound the orthogonal information offered by MS measurements of chemical makeup with atomic- level information afforded by cryo-EM imaging. Further, drawing on concepts from astrophysics already resonant with MS, we anticipate that our highly creative method will overcome pervasive bottlenecks in cryo-EM sample preparation that often limit image resolution. To that end, we have adapted a mass spectrometer to purify and land native protein complexes on cryogenically cooled TEM grids, which are then coated with amorphous ice in vacuo. The resulting grid would represent the ideal cryo-EM sample – a high density of particles situated in random orientations in the same focal plane and covered with only a few nanometers of ice. We plan to achieve even greater structural insight through the incorporation of gas-phase dissociation techniques, which partition the macromolecule into its subcomponents. In particular we propose the addition of surface-induced dissociation (SID) and activated-ion ETD (AI-ETD) to the mass spectrometer used for grid preparation. In short, this technology has genuine potential to create a new paradigm of proteome-scale structural biology.