Radical footprinting in mammalian whole blood

Hydroxyl Radical Protein Footprinting is a powerful tool to probe protein higher-order structure, protein-protein and protein-ligand interactions. It is mostly performed in vitro, but recent advances have extended its use to live cells, nematodes, and 3D cultures. However, to the best of our knowledge, application in living mammalian tissues has not been accomplished. Here, we present the successful use of radical protein footprinting (RPF) in mammalian whole blood from wild-type and type 2 diabetes mellitus (T2DM) mice. Using persulfate photoactivated with the FOX Photolysis System, we achieve effective protein labeling without significant disruption to blood cell morphology. An optimized quenching protocol eliminates background labeling. We report oxidative modifications for the eleven most abundant proteins detected, revealing disease-associated conformational changes in multiple proteins. We validate that in-blood RPF identifies changes in protein structure resulting from complement activation and increased transferrin iron saturation in T2DM mice. These findings demonstrate the feasibility of RPF in mammalian blood and create opportunities for structural proteomics in preclinical models and clinical samples.