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We report a new approach for the fast photochemical oxidation of proteins (FPOP) whereby iodine species are used as the modifying reagent. We generate the radicals by photolysis of iodobenzoic acid at 248 nm; the putative iodine radical then rapidly modifies the target protein. This iodine-radical labeling is sensitive, tunable, and site-specific, modifying only histidine and tyrosine residues in contrast to OH radicals that modify 14 amino-acid side chains. We iodinated myoglobin (Mb) and apomyoglobin (aMb) in their native states and analyzed the outcome by both top-down and bottom-up proteomic strategies. Top-down sequencing selects a certain level (addition of one I, two I's) of modification and determines the major components produced in the modification reaction, whereas bottom-up reveals details for each modification site. Tyr146 is found to be modified for aMb but less so for Mb. His82, His93, and His97 are at least 10 times more modified for aMb than for Mb, in agreement with NMR studies. For carbonic anhydrase and its apo form, there are no significant differences of the modification extents, indicating their similarity in conformation and providing a control for this approach. For lispro insulin, insulin-EDTA, and insulin complexed with zinc, iodination yields are sensitive to differences in insulin oligomerization state. The iodine radical labeling is a promising addition to protein footprinting methods, offering higher specificity and lower reactivity than ∙OH and SO(4)(-∙), two other radicals already employed in FPOP.

New Protein Footprinting: Fast Photochemical Iodination Combined with Top-Down and Bottom-Up Mass Spectrometry