A Fast Photochemical Oxidation of Proteins (FPOP) platform for free-radical reactions: the carbonate radical anion with peptides and proteins.

Fast Photochemical Oxidation of Protein (FPOP), based on a pulsed KrF laser (248 nm) for free-radical generation, is a biophysical method that utilizes hydroxyl radicals to footprint proteins in solution. FPOP has been recognized for structural proteomics investigations, including epitope mapping, protein-aggregation characterization, protein-folding monitoring, and binding-affinity determination. The distinct merits of the platform are: i) the use of a scavenger to control radical lifetime and allow fast ("snapshot") footprinting of solvent-accessible residues in a protein; ii) the employment of a flow system to enable single-shot irradiation of small plugs of the targeted sample; iii) the use of methionine and catalase after radical oxidation chemistry to prevent post-oxidation with residual oxidizing species; and iv) the utilization of mature mass spectrometry-based proteomic methods to afford detailed analysis. In addition to •OH, other reactive reagents (e.g., carbenes, iodide, sulfate radical anion, and trifluoromethyl radical) can be implemented on this platform to increase the versatility and scope. In this study, we further elaborate the use of FPOP platform to generate secondary radicals and establish a workflow to answer fundamental questions regarding the intrinsic selectivity and reactivity of radicals that are important in biology. Carbonate radical anion is the example we chose owing to its oxidative character and important putative pathogenic roles in inflammation. This systematic study with model proteins/peptides gives consistent results with a previous study that evaluated reactivity with free amino acids and shows that methionine and tryptophan are the most reactive residues with CO3-•. Other aromatic amino acids (i.e., tyrosine, histidine and phenylalanine) exhibit moderate reactivity, whereas, aliphatic amino acids are inert, unlike with •OH. The outcome demonstrates this approach to be appropriate for studying the fast reactions of radicals with proteins.