Millisecond radiolytic modification of peptides by synchrotron X-rays identified by mass spectrometry

Radiolysis of peptide and protein solutions with high-energy X-ray beams in duces stable, covalent modifications of amino acid residues that are useful for synchrotron protein footprinting. A series of 5-14 amino acid residue peptides of varied sequences were selected to study their synchrotron radio lysis chemistry. Radiolyzed peptide products were detected within 10 ms of exposure to a white light synchrotron X-ray beam. Mass spectrometry techniq ues were used to characterize radiolytic modification to amino acids cystei ne (Cys), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), proline (Pro), histidine (His), and leucine (Leu). A reactivity ord er of Cys, Met much greater than Phe, Tyr, > Trp > Pro > His, Leu was deter mined under aerobic reaction conditions from MS/MS analysis of the radiolyz ed peptide products. Radiolysis of peptides in O-18-labeled water under aer obic conditions revealed that oxygenated radical species from air and water both contribute to the modification of amino acid side chains. Cysteine an d methionine side chains reacted with hydroxyl radicals generated from radi olysis of water as well as molecular oxygen. Phenylalanine and tyrosine res idues were modified predominantly by hydroxyl radicals, and the source of m odification of proline was exclusively through molecular oxygen.