In-Cell Fast Photochemical Oxidation of Proteins for Proteome Wide Structural Biology

Abstract

Measuring a drug's target engagement in cell is critical for drug discovery. Drug binding studies with purified proteins in vitro may not display interactions that are present in the complex environment of cells. Cellular thermal shift assay (CETSA) has previously been used as an in-cell method to study changes in protein melting temperature due to drug binding. Many proteins, including membrane proteins, are not detectable by CESTA, limiting this method to study drug engagement. To alleviate some of these drawbacks, we proposed to use in-cell fast photochemical oxidation of proteins (IC-FPOP) for Proteome-Wide Structural Biology (PWSB) to study the effect of Gleevec on breast cancer cells. Gleevec was tested on two breast cancer cell lines, triple negative breast cancer (TNBC) from African ancestry (TNBC-AA) and from European ancestry (TNBC-EA), which are both negative for estrogen receptors, progesterone receptors, and human epidermal growth factor receptor. Control cells, which are not treated with Gleevec, and sample cells, which are treated with Gleevec, were subjected to FPOP using a flow system allowing for single cell flow past the laser irradiation and collected in a dimethylthiourea and N-tert-Butyl-a-phenylnitrone (PBN) quench solution. Immediately following FPOP, all cellular proteins were isolated and digested with trypsin. After bottom-up LC-MS/MS, significant changes in FPOP modifications between the control and sample cells were observed proteome wide demonstrating multiple proteins were affected by Gleevec.