TPOP: A Laser-Based, Peroxide-Free Approach for Microsecond Hydroxyl Radical Protein Footprinting in Water

We introduce here a novel protein footprinting approach termed "two-photon oxidation of proteins (TPOP)". This method employs a nanosecond, high-power, 248 nm excimer laser to induce two-photon absorption in both water and aromatic amino acids within proteins. The two-photon absorption of water generates hydroxyl radicals for footprinting, whereas biphotonic excitation of aromatic amino acids produces radical cations that subsequently react with surrounding water to yield hydroxylated products. This photochemistry enables oxidative labeling of protein side chains on the microsecond time scale in pure water, eliminating the need for external oxidants or radical precursors. Importantly, the use of an excimer laser provides a simpler, accessible, efficient, and biocompatible alternative to the existing platforms for the generation of •OH and possibly other radicals. We demonstrated the structural relevance of TPOP labeling using myoglobin as a model protein.