Hydroxyl-Radical Reaction Pathways For The Fast Photochemical Oxidation Of Proteins Platform As Revealed By O-18 Isotopic Labeling

Fast photochemical oxidation of protein (FPOP) has become an important mass spectrometry-based protein footprinting approach. Although the hydroxyl radical ((OH)-O-center dot) generated by photolysis of hydrogen peroxide (H2O2) is most commonly used, the pathways for its reaction with amino-acid side chains remain unclear. Here, we report a systematic study of (OH)-O-center dot oxidative modification of 13 amino acid residues by using O-18 isotopic labeling. The results differentiate three classes of residues on the basis of their oxygen uptake preference toward different oxygen sources. Histidine, arginine, tyrosine, and phenylalanine residues preferentially take oxygen from H2O2. Methionine residues competitively take oxygen from H2O2 and dissolved oxygen (O-2), whereas the remaining residues take oxygen exclusively from O-2. Results reported in this work deepen the understanding of (OH)-O-center dot labeling pathway on a FPOP platform, opening new possibilities for tailoring FPOP conditions in addressing many biological questions in a profound way.