Establishing Signature Fragments for Identification and Sequencing of Dityrosine Cross-Linked Peptides Using Ultraviolet Photodissociation Mass Spectrometry

Dityrosine
cross-linking of Aβ peptides and α-synuclein
is increasingly becoming recognized as a biomarker of neuropathological
diseases. However, there remains a need for the development of analytical
methods that enable the specific and selective identification of dityrosine
cross-linked proteins and peptides in complex biological samples.
Here, we report that the gas-phase fragmentation of protonated dityrosine
cross-linked peptides under ultraviolet photodissociation (UVPD) tandem
mass spectrometry (MS/MS) conditions results in the cleavage across
Cα and Cβ atoms of the dityrosine
residue. This Cα–Cβ cleavage
in UVPD–MS/MS results in the formation of diagnostic pairs
of product ions, providing information on the two individual peptides
involved in the cross-linking, resolving the intrinsic “n2 problem” plaguing the identification
of this post-translational modification (PTM) by tandem mass spectrometry.
Sequencing of a heterodimeric dityrosine cross-linked peptide was
demonstrated using hybrid UVPD–MS/MS and CID–MS3 on a diagnostic pair of product ions. In combination with
dedicated MS–cleavable MSn software,
UVPD–MSn therefore provides an
avenue to selectively discover and describe dityrosine cross-linked
peptides. Additionally, observation of dityrosine-specific “reporter
ions” at m/z 240.1019 and m/z 223.0752 in UVPD–MS/MS will
be useful for the validation of the dityrosine cross-linked peptides.