Identification and relative quantification of 3-nitrotyrosine residues in fibrinogen nitrated in vitro and fibrinogen from ischemic stroke patient plasma using LC-MS/MS

Romina Medeiros, Bebiana Sousa, Silvina Rossi, Catarina Afonso, Luis Bonino, Andrew Pitt, Elizabeth López, Corinne Spickett, Graciela Borthagaray

Research output: Contribution to journalArticlepeer-review

Abstract

Ischemic stroke is one of the leading causes of death and disability worldwide. This acute vascular event interferes with blood supply to the brain and induces a burst of free radicals such as nitric oxide and superoxide, producing peroxynitrite, a precursor of strong nitrating agents. Fibrinogen is one of the most abundant plasma proteins; it plays a role in the hemostatic system, mediating clot formation, which can be affected by nitrotyrosine formation. We hypothesized that nitration of fibrinogen by ONOOH and ONOOCO radical products could be one of the early events of the ischemic stroke, and protein-bound 3-nitrotyrosine could be a potential biomarker for diagnosis and/or prognosis of this condition. A targeted mass spectrometry approach was developed to analyze the nitration of fibrinogen and its association with ischemic stroke. First, a comprehensive mapping of 3-nitrotyrosine locations and their relative quantification was performed by LC-MS/MS, using in vitro nitrated fibrinogen samples. Twenty different 3-nitrotyrosine residues were identified on fibrinogen nitrated in vitro, varying with the peroxynitrite tofibrinogen molar ratio used. Nine tyrosine residues that were consistently modified at different treatment ratios were chosen to perform a targeted LC-MS/MS analysis in clinical samples. Enriched fibrinogen fractions from clinical samples from 24 ischemic stroke and 12 patients with non-inflammatory conditions were analysed with this method. Three of the nine tyrosine residues analysed (βY452, βY475 and γY380) showed a significant difference between the ischemic stroke and non-inflammatory disease groups. ROC curve analysis suggested an association of these residues either individually or in combination with ischemic stroke. Different tyrosine nitration patterns were also observed in fibrinogen modified in vitro and in vivo, suggesting differences in the nitration process in these situations. This is the first study showing a putative association between the nitration profile of specific tyrosine residues in human fibrinogen and ischemic stroke. [Abstract copyright: Copyright © 2021 Elsevier Inc. All rights reserved.]
Original languageEnglish
Pages (from-to)334-347
Number of pages14
JournalFree Radical Biology and Medicine
Volume165
Early online date4 Feb 2021
DOIs
Publication statusPublished - 1 Mar 2021

Bibliographical note

© 2021, Elsevier. Licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/

This project has received funding from Comisión Sectorial de Investigación Científica (CSIC), from PEDECIBA-QUÍMICA, and from the European Union's Horizon 2020 research and innovation programme under the Marie Sklowdowska-Curie grant agreement number 675132 www.masstrplan.org.

Keywords

  • Biomarkers
  • Diagnostic methods
  • Inflammation
  • Nitroxidative stress
  • Oxidative post-translational modification
  • ROC curve Analysis
  • Tandem mass spectrometry

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