Hydroxyl radical-mediated modification of proteins as probes for structural proteomics.

ion of hydrogen from the γ-(C-4) carbon of aliphatic side chains in the presence of oxygen can yield C-2 -C-3 dehydropeptides, via the formation of peroxyl radicals from the γ-carbon-centered radical. 208,210 For example, in the oxidation of the Glu side chain, the γ-carbon peroxyl radical may undergo subsequent reactions leading to the formation of side chain modification products with a mass shift of -30 Da due to decarboxylation; 209,211+14 and+16 Da products; or an unsaturated product, a C-2 -C-3 dehydropeptide (Scheme 4). The dehydropeptide behaves like an oxygenated enol species, which readily undergoes tautomerism to the keto form. This species is easily hydrolyzed to yield two protein fragments: a new amide and a keto acid. 210 Oxidation of aspartic acid seems similar to the case of Glu and may also result in the cleavage of the protein backbone 212 or decarboxylation of a side chain carboxyl group, 211 except at a lower rate than that for Glu. 211 3.2.4. Main Chain Cleavage via Radical Transfer from the â-Carbon at Side Chains Initial hydroxyl radical attack at theâ-carbon (C-3) position can lead to the formation of R-carbon radicals and subsequent main chain rupture. 213,214 In this case, alkoxyl radicals are generated after initial H-abstraction to produce carbon-centered radicals and subsequent reaction with O 2 o give peroxyl radicals (Scheme 5). A number of different pathways may generate alkoxyl radical from different precursors, including termination reactions of the peroxyl species with other radicals and decomposition of the intermediate hydroperoxides. 215,216The alkoxyl radicals can undergoâ-scission at a rate >107 s-1, leading to the loss of a side chain and/or generation of R-carbon radicals and subsequent backbone cleavage. 213,214 The â-scission reaction of alkoxyl radicals appears to be common for aliphatic side chains such as Val, Leu, and Asp,214 resulting in the release of a family of carbonyls including formaldehyde, acetone, isobutyraldehyde, and glyoxylic acids. The rate of suchâ-scission reactions is affected by the nature of the substituents, R and R ′, with the rate of fragmentation increased by the presence of electron releasing alkyl groups and substituents that can stabilize the incipient radical center. The R-carbon radicals generated by the â-scission reaction of alkoxyl radicals are stable due to the delocalization of unpaired electrons onto the neighboring carbonyl and amide functions and relief of steric strain in the alkoxyl radical. 217 Scheme 3. Backbone Cleavage and Side Chain Modification of Pro Hydroxyl Radical-Mediated Modification of Proteins Chemical Reviews, 2007, Vol. 107, No. 8 3527