Characterization and Identification of Dityrosine Cross-Linked Peptides Using Tandem Mass Spectrometry

The use of mass spectrometry coupled with Chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or Cysteine residues, identification of gas-phase fragmentation patterns of endogenus dityrosine Cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress,, aging, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked driller of A beta(1-16), using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked A beta(1-16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of A beta and alpha-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and alpha-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions.