Validation of the Applicability of In-Cell Fast Photochemical Oxidation of Proteins across Multiple Eukaryotic Cell Lines

Fast
photochemical oxidation of proteins (FPOP), a hydroxyl radical-based
protein footprinting method, coupled to mass spectrometry has been
extensively used to study protein structure and protein–protein
interactions in vitro. This method utilizes hydroxyl
radicals to oxidatively modify solvent-accessible amino acids and
has recently been demonstrated to modify proteins within live cells
(IC-FPOP) and Caenorhabditis elegans. Here, we have
expanded the application of IC-FPOP into a variety of commonly used
cell lines to verify the applicability of the method across various
cellular systems. IC-FPOP was able to successfully modify proteins
in five different cell lines (Vero, HEK 293T, CHO, MCF-10A, and MCF-7).
To increase the number of oxidatively modified proteins identified,
we have also employed the use of offline high pH reversed-phase liquid
chromatography (RPLC) followed by concatenation and online low-pH
RPLC. The coupling of IC-FPOP to 2D-LC MS/MS resulted in a 1.7-fold
increase in total identifications of oxidatively modified proteins,
which expanded the dynamic range of the method. This work demonstrates
the efficacy of using IC-FPOP to study protein–protein interactions
in cells.