Establishing signature fragments for identification and sequencing of Dityrosine cross-linked peptides using ultraviolet photodissociation mass spectrometry

Abstract

Dityrosine cross-linking of A beta peptides and alpha-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 C-alpha and C-beta atoms of the dityrosine residue. This C-alpha-C-beta 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 "n(2) 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 crosslinked 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.