Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Drăguşanu, Mihaela; Petre, Brînduşa-Alina; Przybylski, Michael

Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry

Dateien

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Datum

2011

Autor:innen

Drăguşanu, Mihaela

Petre, Brînduşa-Alina

Przybylski, Michael

Publikationstyp

Zeitschriftenartikel

unikn.publication.listelement.citation.prefix.version.undefined

Journal of Peptide Science. 2011, 17(1), pp. 32-38. ISSN 1075-2617. eISSN 1099-1387. Available under: doi: 10.1002/psc.1293

Zusammenfassung

Nitration of tyrosine residues has been shown to be an important oxidativemodification in proteins and has been suggested to AQ1 play a role in several diseases such as atherosclerosis, asthma, and lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine-specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified byMS.Wehave used amonoclonal 3-NT-specific antibody, and have synthesized a series of tyrosine-nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr-430 had been previously identified upon reaction with peroxynitrite [17]. The determination of antibody-binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr-430, Tyr-421, Tyr-83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N-terminal to the nitration site. The highest affinity to the anti-3NT-antibody was found for the PCS peptide comprising the Tyr-430 nitration site with a KD of 60 nM determined for the peptide, PCS(424-436-Tyr-430NO2); in contrast, PCS peptides nitrated at Tyr-421 and Tyr-83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody-bound peptides and affinity-MS analysis revealed highest affinity to the antibody for tyrosine-nitrated peptides that contained positively charged amino acids in the N-terminal sequence to the nitration site. Remarkably, similar N-terminal sequences of tyrosine-nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil-cationic protein.

Fachgebiet (DDC)

540 Chemie

Schlagwörter

tyrosine nitration, affinity-mass spectrometry approaches, proteolytic epitope excision, 3-nitro-tyrosine antigen-antibody complex

Zitieren

ISO 690

DRĂGUŞANU, Mihaela, Brînduşa-Alina PETRE, Michael PRZYBYLSKI, 2011. Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry. In: Journal of Peptide Science. 2011, 17(1), pp. 32-38. ISSN 1075-2617. eISSN 1099-1387. Available under: doi: 10.1002/psc.1293

BibTex

@article{Dragusanu2011-01Epito-1014,
  year={2011},
  doi={10.1002/psc.1293},
  title={Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry},
  number={1},
  volume={17},
  issn={1075-2617},
  journal={Journal of Peptide Science},
  pages={32--38},
  author={Drăguşanu, Mihaela and Petre, Brînduşa-Alina and Przybylski, Michael}
}

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