Molecular Recognition Specificity of anti-3-nitrotyrosine Antibodies Revealed by Affinity-Mass Spectrometry and Immunoanalytical Methods

Abstract

Nitration of tyrosine residues in proteins has been mainly characterised by immunoanalytical methods using anti-3-nitrotyrosine antibodies, and nitration sites and sequences have been hitherto identified only in a few cases using mass spectrometric methods. Immuno-analytical methods frequently suffer from low and poorly characterised detection specificity of antinitrotyrosine antibodies, while mass spectrometric methods for identification of Tyrosine nitration may be hampered by low levels of modification, and by possible changes of structure and proteolytic degradation of proteins introduced by the nitration. Moreover, no detailed, molecular characterisation of the specificity of anti-3-nitrotyrosine antibodies has been reported. In this study we describe a molecular study of the recognition specificities and affinities of two commercially available, monoclonal anti-nitrotyrosine antibodies by affinity-mass spectrometry, using different 3-nitrotyrosine containing peptides. Tyrosine-nitrated and non-nitrated substrate peptides of prostacyclin synthase (PCS), an enzyme inactivated by nitration of the active site Tyr-430 residue, were synthesised by solid-phase peptide synthesis (SPPS), purified by reversed phasehigh performance liquid chromatography (RP-HPLC) and characterised by electrospray (ESI) and matrix-assisted laser desorption-ionisation (MALDI) mass spectrometry. Binding affinities and specificities of PCS peptides with different Tyr-nitration sites and sequence mutations adjacent to Tyr-430 were determined by evaluation of anti-nitrotyrosine antibodies using an affinitymass spectrometry approach, compared to immuno-analytical determination using dot-blot and ELISA. The results showed that the antibodies may discriminate in the recognition of peptides with different N-terminal adjacent sequences to the nitrotyrosine residues, depending on the type of immunogen employed. A quantitative ELISA estimation was developed for the determination of antibody binding by Tyrosine-nitrated peptides.