Implementing In-Cell Fast Photochemical Oxidation of Proteins in a Platform Incubator with a Movable XY Stage.

Abstract

Fast photochemical oxidation of proteins (FPOP) is a protein footprinting technique that is being increasingly used in MS-based proteomics. FPOP is utilized to study protein–protein interactions, protein–ligand interactions, and protein conformational dynamics. This method has recently been extended to protein labeling in live cells (IC-FPOP), allowing the study of protein conformations in the complex cellular environment. Traditionally, IC-FPOP has been executed using a single cell flow system, in which hydrodynamic focusing drives cells along in a single file line, keeping the cells from clumping and thus ensuring equal exposure to the laser irradiation required for photochemical oxidation. Here, we introduce a novel platform that allows IC-FPOP to occur in a sterile incubation system complete with a mobile stage for XY movement, peristaltic pumps equipped with perfusion lines for chemical transport, and mirrors for laser beam guidance. This new system, called Platform Incubator with movable XY stage (PIXY), also utilizes software enabling automated communication between equipment and execution of the entire system. Further, comparison with a standard IC-FPOP flow system results reveal that this platform can successfully be used in lieu of the flow system while also decreasing the time to complete analysis of a single sample.