Onset of oxidative damage in α-crystallin by radical probe mass spectrometry

The early onset oxidative damage within segments of the protein α-crystallin is examined by radical probe mass spectrometry by its treatment with reactive oxygen species under low-, moderate-, and high-oxidizing conditions. Five regions comprising the first 11 residues of the N-termini of the A and B subunits (A/B:1-11), central domains from each subunit B:57-69 and A:104-112, and a C-terminal segment of the A subunit A:120-145 were found to be the initial sites of oxidation. The susceptibility of each segment to oxidation and oxidative damage is investigated by subjecting the intact protein to different oxidation conditions within the ion source of an electrospray ionization mass spectrometer. LC-MS of the oxidized protein digests enables the sites and levels of oxidation to be monitored. The onset of oxidative damage and the levels of oxidation observed before damage occurs differ across the protein surface. The regions comprising residues A/B:1-11 and A:104-112 are shown to be more susceptible to oxidative damage than those comprising residues B:57-69 and A:120-145. The results are discussed in the context of available experimental and homology-modeled theoretical structures for the subunits of α-crystallin.