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Advanced methodology combining UPLC-MS, isotopic labelling and H/D exchanges reveals three tyrosine-tyrosine cross-links induced by oxidative radicals evolving to at least four dimeric structures

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Abstract

Di-tyrosine is one of the major protein cross-links involved in a large number of neurodegenerative or ageing-related diseases. Recently, no less than four different di-tyrosine bridge isomers have been highlighted while only two structures are characterized at the moment in the literature. In this study, the four dimers were produced by radiolytical-induced oxidation. Although the abundance of these additional dimers precluded the use of NMR or other structural characterization methods, we propose a new methodology combining UPLC-MS analysis, specific deuterium labelling and isotopic (H/D) exchanges with the solvent. Thus, we were able to identify three different covalent cross-links and propose different new original di-tyrosine structures based on double Michael additions, leading to tetracyclic products. Absorption and fluorescence characterizations of the four species were performed and consolidate our proposal.

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Abbreviations

DOPA:

Dihydroxyphenylalanine

FLD:

Fluorescence detection

H/D:

Hydrogen/deuterium

HOHICA:

3A-Hydroxy-6-oxo-2,3,3a,6,7,7a-hexa-hydro-1H-indole-2-carboxylic acids

HPLC:

High-performance liquid chromatography

MAD:

Michael addition dimer

MS:

Mass spectrometry

NMR:

Nuclear magnetic resonance

NOE:

Nuclear Overhauser effect

PDA:

Photo diode array detector

UPLC:

Ultra high-performance liquid chromatography

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Funding

This research did not receive any direct specific grant from funding agencies in the public, commercial or not-for-profit sectors. Access to the UPLC-MS was funded as the MOBICS project funded by a DIM Analytics programme from the Ile-de-France region.

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Authors and Affiliations

  1. CNRS, Institut de Chimie Physique UMR 8000, Université Paris-Saclay, 91405, Orsay Cedex, France

    Isabelle Billault, Anouchka Gatin, Guillaume Van der Rest & Cécile Sicard-Roselli

Authors
  1. Isabelle Billault
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  2. Anouchka Gatin
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  3. Guillaume Van der Rest
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  4. Cécile Sicard-Roselli
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Contributions

Experiments were performed by I. Billault, A. Gatin and C. Sicard-Roselli. The manuscript was written by I. Billault, A. Gatin C. Sicard-Roselli and G. Van der Rest.

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Correspondence to Cécile Sicard-Roselli.

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Billault, I., Gatin, A., Van der Rest, G. et al. Advanced methodology combining UPLC-MS, isotopic labelling and H/D exchanges reveals three tyrosine-tyrosine cross-links induced by oxidative radicals evolving to at least four dimeric structures. Anal Bioanal Chem 414, 1595–1607 (2022). https://doi.org/10.1007/s00216-021-03782-x

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  • DOI: https://doi.org/10.1007/s00216-021-03782-x

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