Fast Photochemical Oxidation of Protein Footprints Faster than Protein Unfolding

Abstract

Fast photochemical oxidation of proteins (FPOP) is a chemical footprinting method whereby exposed amino-acid residues are covalently labeled by oxidation with hydroxyl radicals produced by the photolysis of hydrogen peroxide. Modified residues can be detected by standard trypsin proteolysis followed by LC/MS/MS, providing information about solvent accessibility at the peptide and even the amino-acid level. Like other chemical footprinting techniques, FPOP must ensure only the native conformation is labeled. Although oxidation via hydroxyl radical induces unfolding in proteins on a timescale of milliseconds or longer, FPOP is designed to limit •OH exposure to 1 μs or less by employing a pulsed laser for initiation to produce the radicals and a radical-scavenger to limit their lifetimes. We applied FPOP to three oxidation-sensitive proteins and found that the distribution of modification (oxidation) states is Poisson when a scavenger is present, consistent with a single conformation protein modification model. This model breaks down when a scavenger is not used and/or hydrogen peroxide is not removed following photolysis. The outcome verifies that FPOP occurs on a time scale faster than conformational changes in these proteins.

Probing the Time Scale of FPOP (Fast Photochemical Oxidation of Proteins): Radical Reactions Extend Over Tens of Milliseconds

“…The ⋅OH concentration profile calculated under pseudo-first order conditions for [Gln] = 20 mM drops close to zero within 1 μs (blue line, Figure 1b) [30]. This plot represents the foundation of the claim that FPOP is a microsecond covalent labeling technique [30][31][32].…”

“…The acronym FPOP (fast photochemical oxidation of proteins) has been coined for this technique [31]. It was claimed that FPOP goes to completion within~1 μs.…”

“…This extraordinarily short time frame has important implications. First, under single-hit conditions, FPOP should be immune to labeling-induced artifacts because protein tagging will be complete before conformational changes can take place [30][31][32]. Secondly, FPOP holds promise for examining short-lived protein folding intermediates [33].…”

“…Instead, the assertion of a~1 μs labeling pulse is based on kinetic estimates [30], indirect statistical data [31], and stationary (not timeresolved) dosimetry [32]. It is instructive to revisit the key arguments [30].…”

“…It cannot be ruled out that secondary radicals contribute to analyte oxidation within the FPOP flow tube. In other words, the claim that FPOP labeling is restricted to ã 1 μs time scale [30][31][32] has to be carefully re-examined.…”