Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins.

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids.

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.

An efficient quantitation strategy for hydroxyl radical-mediated protein footprinting using Proteome Discoverer.

Hydroxyl radical protein footprinting coupled with mass spectrometry has become an invaluable technique for protein structural characterization. In this method, hydroxyl radicals react with solvent exposed amino acid side chains producing stable, covalently attached labels. Although this technique yields beneficial information, the extensive list of known oxidation products produced make the identification and quantitation process considerably complex.

Fast photochemical oxidation of proteins coupled to multidimensional protein identification technology (MudPIT): expanding footprinting strategies to complex systems.

Peptides containing the oxidation products of hydroxyl radical-mediated protein footprinting experiments are typically much less abundant than their unoxidized counterparts. This is inherent to the design of the experiment as excessive oxidation may lead to undesired conformational changes or unfolding of the protein, skewing the results. Thus, as the complexity of the systems studied using this method expands, the detection and identification of these oxidized species can be increasingly difficult with the limitations of data-dependent acquisition (DDA) and one-dimensional chromatography.