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

J Pept Sci. 2011 Mar;17(3):184-91. doi: 10.1002/psc.1298. Epub 2011 Jan 11.


Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, 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 by MS. We have used a monoclonal 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 peroxynitrite17. 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 K(D) of 60 nM determined for the peptide, PCS(424-436-Tyr-430NO(2) ); 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies / chemistry*
  • Antibodies / immunology*
  • Chromatography, High Pressure Liquid
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / chemistry*
  • Epitopes / immunology
  • Mass Spectrometry
  • Molecular Sequence Data
  • Peptides / immunology*
  • Tyrosine / analogs & derivatives*
  • Tyrosine / chemistry
  • Tyrosine / immunology


  • Antibodies
  • Epitopes
  • Peptides
  • 3-nitrotyrosine
  • Tyrosine