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

Abstract

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 C α and C β atoms of the dityrosine residue. This C α −C β 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−MS 3 on a diagnostic pair of product ions. In combination with dedicated MS−cleavable MS n software, UVPD−MS n 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.

Characterization of Insulin Dimers by Top-Down Mass Spectrometry

“…The contribution of these four aromatic side chains in the two cross-linked Phe1-His5 regions in the presence of eight peptide bonds increased the total extinction coefficient 2.8-fold at 214 nm. Excitation of Phe and His may thereby have contributed to the efficient sequence coverage with 213 nm UVPD, as energy transfer can also lead to fragmentation. , Also, a recent study reported 213 nm UVPD of diTyr cross-linked peptide dimers that in a similar manner to our observations led to separation of the monomers by fragmentation of the C α -C β bond in the chromophoric cross-link . Fragmentation of the C α -C β bond induced by UVPD has also been reported for Phe, Tyr, and Trp residues. − Thus, the confident sequence coverage and detailed fragmentation of the cross-link in the 2M+C dimer produced by 213 nm UVPD may have been significantly improved by the chromophoric capacity of the Phe and His side chains.…”

“…38,53 Also, a recent study reported 213 nm UVPD of diTyr cross-linked peptide dimers that in a similar manner to our observations led to separation of the monomers by fragmentation of the C α -C β bond in the chromophoric cross-link. 54 Fragmentation of the C α -C β bond induced by UVPD has also been reported for Phe, Tyr, and Trp residues. 55−57 Thus, the confident sequence coverage and detailed fragmentation of the cross-link in the 2M+C dimer produced by 213 nm UVPD may have been significantly improved by the chromophoric capacity of the Phe and His side chains.…”

Photosensitized Oxidative Dimerization at Tyrosine by a Water‐Soluble 4‐Amino‐1,8‐naphthalimide

“…Following purification by reverse-phase HPLC (RP-HPLC), synthetic 7 was characterized by 1 H and 13 C nuclear magnetic resonance (NMR) spectroscopy and direct injection mass spectrometry with electrospray ionization (DIMS-ESI), and the spectra were found to be in agreement with previous studies. [11,40,51] With 7 as a comparative standard, conditions for the photosensitized oxidative dimerization of Ac-Tyr-OH (6) were evaluated using 1. In a preliminary screening, it was found that visible-light irradiation (blue LEDs, peak wavelength: 445 nm; radiant flux: 60 W; irradiance: 50 mW/cm 2 ) of 1 in 100 mM sodium phosphate buffer (PB) solution at pH = 7.0 effectively promoted the oxidative dimerization of 6 to provide dityrosine 7.…”

“…Related aryl-derived linkages can also be formed following photooxidation of the electron-rich amino acid residues histidine (His), phenylalanine (Phe), tryptophan (Trp), and Tyr upon protein exposure to sunlight. [10][11][12][13][14] Photooxidation reactions can occur directly by incident UVB radiation absorption at a residue chromophore or indirectly as mediated by a photosensitizer. Specifically, covalently-linked dityrosine moieties (B) are targeted by studies aimed at understanding how oxidation-induced cross-links are formed and how their presence can alter the physical properties of a system.…”

Mass Spectral Signatures of Complex Post-Translational Modifications in Proteins: A Proof-of-Principle Based on X-ray Irradiated Vancomycin

“…A combination of electron-transfer dissociation and CID has been applied for studying cyclotides containing numerous cysteine knots . VUV photoionization has recently been proposed for the identification of multiple PTMs in histones. , In 2019, photodissociation using a UV laser revealed mass spectral signatures of dityrosine cross-linking in peptide dimers …”