title: Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry.
abstract: 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.
authors.affiliation: Laboratory of Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry, University of Konstanz, D-78457 Konstanz, Germany.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Dr Guşanu
authors.forename: Brînduşa-Alina
authors.initials: BA
authors.lastname: Petre
authors.forename: Michael
authors.initials: M
authors.lastname: Przybylski
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry* / Q000276:immunology*; D002851:Chromatography, High Pressure Liquid; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology*; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology
chemical_list: D000906:Antibodies; D000939:Epitopes; D010455:Peptides; C002744:3-nitrotyrosine; D014443:Tyrosine