Improved High-Throughput Platform for In-Cell Fast Photochemical Oxidation of Proteins via an Automated XY Stage (AXYS).

Mass spectrometry (MS)-based protein footprinting and, more specifically, fast photochemical oxidation of proteins (FPOP) are methods that have been found to be important for studying proteins, their structures, and their relationships to other proteins or ligands. In-cell FPOP (IC-FPOP) was developed to study proteins in their native environment. Initial work with IC-FPOP has been performed using a platform incubator with an XY movable stage (PIXY). However, low throughput and a six-well plate format restricted the experiment by limiting the number of technical replicates that can be analyzed at one time and requiring large amounts of samples per experiment. Here, we introduce an improved, higher throughput platform that allows IC-FPOP to be run on a fully automated XY stage (AXYS) using 24-well plates. Comparison with the PIXY system results shows that this platform can successfully modify more proteins in less time. AXYS also increases the types of biological samples that can be analyzed by IC-FPOP.