Fast Photochemical Oxidation of Proteins (FPOP)

photochemical oxidation

Photochemical lablelling of proteins is one of the structural proteomics approaches being employed by the Facility to probe solution phase structure of proteins. Mass spectrometry is used to map sites of covalent chemical modification to examine solvent accessiblity and how this changes under different conditions. This technique can be used to investigate conformational changes of a protein or protein complexes in the presence or absence of a small molecule for example or to determine the binding interfaces between individual proteins in a higher order complex.


Fast photochemical oxidation of proteins uses a solution of proteins in the presence of small amounts of hydrogen peroxide and radial scavenger glutamine. When the solution is exposed to bursts of laser irradiation, the hydrogen peroxide breaks down to form hydroxyl radicals that quickly react with amino acid side chains on the proteins causing oxidation. The speed of the oxidation reaction is much faster than any conformational change that may occur and the presence of the glutamine ensures a short lifetime of the hydroxyl radicals. The position of the oxidations introduced to the molecules are then determined using a bottom-up proteomics approach. Comparison of the levels of oxidation on the peptides under different conditions enables information about solvent accessibility to be inferred.