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

Area of Science:

• Biochemistry
• Analytical Chemistry
• Proteomics

Background:

• Dityrosine cross-linking is a marker for oxidative stress, aging, and neurodegenerative diseases like Alzheimer's and Parkinson's.
• Gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides remain largely uncharacterized.
• Existing methods for protein structure analysis rely on reagents specific to lysine or cysteine residues.

Purpose of the Study:

• To investigate and characterize the fragmentation patterns of dityrosine cross-linked peptides.
• To establish generic fragmentation rules for identifying dityrosine cross-links.
• To apply these rules for detecting dityrosine cross-links in disease-relevant proteins.

Main Methods:

• Synthetically prepared dityrosine cross-linked dimer of Aβ(1-16) was analyzed using ESI tandem mass spectrometry.
• Fragmentation patterns were characterized using Collision Induced Dissociation (CID), Higher-Energy Collision Induced Dissociation (HCD), Electron Transfer Dissociation (ETD), and Electron Capture Dissociation (ECD).
• In vitro enzymatic peroxidation was used to generate dityrosine cross-links in peptides of Aβ and α-synuclein.

Main Results:

• Detailed fragmentation patterns for dityrosine cross-linked Aβ(1-16) were elucidated using various dissociation techniques.
• Generic fragmentation rules for dityrosine cross-linked peptides were established.
• Dityrosine cross-links were identified in Aβ and α-synuclein peptides generated in vitro.
• Dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions were reported for the first time.

Conclusions:

• The established fragmentation rules facilitate the identification of dityrosine cross-links in peptides.
• This methodology enables the detection of a naturally occurring post-translational modification linked to neurodegenerative diseases.
• The findings pave the way for automated analysis of dityrosine cross-links in pathological conditions.