Development of a Microflow System for In-Cell Footprinting Coupled with Mass Spectrometry.

Fast photochemical oxidation of proteins (FPOP) has become a valuable tool for protein structural characterization. The method has recently been demonstrated to oxidatively modify solvent-accessible sites of proteins inside live cells (IC-FPOP). However, the flow system used for in vitro analysis is not well-suited for IC-FPOP as a number of factors can lead to cell aggregation, causing inconsistent labeling and clogging. Here, we present an IC-FPOP flow system that centrally focuses the cells, ensuring consistent radiation exposure. Fluorescence imaging was used to analyze the effectiveness of the system in focusing the cells. Analysis shows the cells flowing individually through the center of the capillary with the buffer visible along the walls and with no aggregation or clogging observed. To ensure the flow system does not disturb oxidative modification, Vero cells were labeled using the flow system and analyzed by liquid chromatography-mass spectrometry (LC-MS). The results demonstrate a 13-fold increase in the number of oxidized proteins and a 2 orders of magnitude increase in the dynamic range of the method.