The Development of In-Cell Protein Footprinting Coupled with Mass Spectrometry for Structural Biology in Complex Model Systems

In recent years, protein footprinting coupled with mass spectrometry has been used to identify protein-protein interaction sites and regions of conformational change through modification of solvent accessible sites in proteins. The footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to modify these solvent accessible sites. To date, FPOP has been used in vitro on relatively pure protein systems. We have further extended the FPOP method for in vivo analysis of proteins. This will allow for study of proteins in their native cellular environment and be especially useful for the study of membrane proteins which can be difficult to purify for in vitro studies. A major application of the in vivo method is for proteome-wide structural biology. To this end, we have further developed the method for patient samples, specifically peripheral blood mononuclear cells (PBMCs). We have optimized several parameters for labeling of proteins in these patient samples. This method have the potential to become a powerful tool in the structural biology toolbox.