Expanding the Use of FPOP for In Vitro and In Cell Studies

Abstract

Studying higher order protein structure is crucial to better understand protein interactions and functions. Mass spectrometry (MS) has been an invaluable tool to better understand protein structure. Several techniques, like protein footprinting, are coupled with mass spectrometry to gain information on protein structure and changes in protein-protein and protein-ligand interactions. Hydroxyl radical protein footprinting (HRPF) utilizes hydroxyl radicals to irreversibly label solvent-exposed side chains of 19 out of the 20 amino acids. Traditionally, modified regions are detected using bottom-up proteomics and with MS/MS analysis, residue level information can be obtained. There are a handful of techniques to generate hydroxyl radicals, one is fast photochemical oxidation of proteins (FPOP). FPOP generates hydroxyl radicals through the photolysis of hydrogen peroxide using a 248 nm excimer laser. My research aims to expand the use of FPOP for in vitro and in cell studies. For in-vitro FPOP the first objective was to validate the use of FPOP to study proteins in their native structure. In all protein footprinting techniques, it is crucial to label while the protein remains in its native structure and ensure labeling does not take place if the protein begins to unfold. To confirm FPOP probed proteins in their native state, the enzymatic activity of proteins were measured before and after FPOP. The second objective was to combine FPOP with native MS, ion mobility separation (IMS), and top-down proteomics to gain additive structural information. Next, the use of in-cell FPOP (IC-FPOP) as a tool for proteome wide structural biology (PWSB) was expanded to characterize drug interactions in cells. First, IC-FPOP was used to highlight the differing effect of Gleevec in triple negative breast cancer (TNBC) for a European ancestry (TNBC-EA) and African ancestry (TNBC-AA) cell line. Finally, IC-FPOP efficacy in probing methotrexate’s effects in a chronic myelogenous leukemia (CML) was compared to cellular thermal shift assay (CETSA). CETSA is a recognized method that can be used to track drug interactions across the full proteome. However, there are some limitations to using CETSA which IC-FPOP can help overcome thus improving the characterization of drug interactions in cells.