Raquel Shortt

Hydroxyl radical protein footprinting (HRPF), a non-specific covalent label (CL), has become a critical component in structural biology studies, as it proved useful in delineating protein structure, protein-protein interactions, and protein conformations. Fast Photochemical oxidation of protein (FPOP), a valuable HRPF technique has recently been extended to the study of intact cells (IC-FPOP) and in an in-vivo system (IV-FPOP). Each labeling method has been shown to modify thousands of proteins in a single experiment for proteome-wide structural biology. This was further demonstrated when IC-FPOP was performed on a platform incubator with an X Y movable stage (PIXY), which modified hundreds of proteins in live cells in a fraction of the time compared to the original flow system. Although IC- and IV-FPOP can reveal proteome-wide structural information, the experimental procedures require tedious sample processing followed by an extensive data analysis workflow post-LC-MS/MS analysis to calculate the extent of modification at the peptide and residue level. Implementation of automated sample handling robots is increasing to avoid the laborious preparation of MS samples. Meanwhile, free open-source, reusable, and customizable coding scripts for the analysis of proteome-wide structural are dismal. Therefore, to expand the knowledge gained from examining the dynamics of an entire proteome from a structural perspective it is important to increase the efficiency of the IC-FPOP workflow and increase accessibility to open-source data automation tools.  In my research, I integrate automated solutions into the IC-FPOP workflow to expedite processes like sample handling and data processing.