In recent years, protein footprinting coupled with mass spectrometry has been used to identify protein-protein interaction sites and regions of conformational change through modification of solvent accessible sites in proteins. The footprinting method, fast photochemical oxidation of proteins (FPOP), utilizes hydroxyl radicals to modify these solvent accessible sites. To date, FPOP has been used in vitro on relatively pure protein systems. We have further extended the FPOP method for in vivo analysis of proteins. This will allow for study of proteins in their native cellular environment and be especially useful for the study of membrane proteins which can be difficult to purify for in vitro studies. Here, we report the use of in-cell FPOP (IC-FPOP) to identify on and off targets of the anti-cancer drug Gleevec. Comparison of oxidative labeling of proteins in drug treated and non-treated cells demonstrates structural changes that occur in response to drug treatment. We found 92 proteins whose level of oxidation changed in response to drug treatment. Many of these proteins are targets of BCR-ABL but we did identify some off targets that are not known to interact with BCR-ABL. This work demonstrates the ability of IC-FPOP to study protein interactions across the proteome.

Identifying protein interactions using in-cell protein footprinting coupled with mass spectrometry

2019 Fall National Meeting

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