Project Summary Mass spectrometry (MS) based footprinting is emerging as a powerful means to answer biological questions about membrane proteins (MPs), which participate in almost all physiological processes and represent more than 60% of drug targets. This approach affords sufficient structural information for the dynamic, native conformations and interactions of MPs in cells, which are beyond the reach of traditional structural methods (e.g., cryo-EM and crystallography). This bottom-up MS footprinting is complementary to but potentially more informative than top-down native MS, which does not provide spatial resolution for MPs and is conducted in the nonnative gas phase. Here we propose to continue development of novel MS footprinting methods in live cells and native membranes. Our objective is to design, prepare, test, and improve chemical probes that provide high footprinting coverage. We will then apply them to reveal drug interactions and cellular trafficking regulation of a glucose transporter, GLUT1, a prominent anticancer drug target and a model MP representing ~ 25% of known transport proteins. MS footprinting of MPs, however, poses three major challenges. 1) MPs, which are hydrophobic and buried in lipid bilayers, are resistant to traditional probes (e.g., HDX, •OH radicals) that penetrate poorly and give insufficient labeling. 2) Aliphatic side chains of transmembrane regions contain C–H and C-C bonds that are unreactive with most chemical probes. 3) The footprinting needs to be conducted in cells or membranes to maintain native conformation and interaction of MPs. Our hypotheses are: (1) Complementary modifications of C-H and X–H bonds by free radicals produced photochemically and by nucleophilic reagents maximize footprinting coverage. (2) Tuning the hydrophobicity of the reagents or their precursors allows access to membrane-embedded MPs. (3) Novel membrane fusion techniques introduce inert footprinters into live cells and native membranes for subsequent photoactivated footprinting. Our hypotheses are built on extensive preliminary data. Three years of funding supported publication of 18 papers in high-profile journals. A significant example describes laser activation of TiO2 nanoparticles attached to liposomes to generate high local concentrations of radicals. Simultaneous membrane poration permits radical entry to footprint with sufficient structural resolution that reports the ligand-binding sites and rocker-switch motions of GLUT1. Building on these successes, we will pursue two specific aims: (1) develop new chemical probes for MS footprinting of MPs; and (2) conduct comprehensive footprinting in native membranes and live cells to reveal anticancer drug interactions and trafficking regulations of GLUT1. Our innovative footprinting coupled with bottom-up MS proteomics analysis will establish bio-orthogonal footprinters that afford comprehensive coverage of both hydrophobic and hydrophilic regions of MPs and reveal drug interac...