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.

When is Mass Spectrometry Combined with Affinity Approaches Essential? A Case Study of Tyrosine Nitration in Proteins

“…The principle of this approach is analogous to a general, previously developed epitope extraction-MS method for identification of antibody-epitopes, in which a protein antigen is digested in solution and the proteolytic peptide mixture presented to the affinity column [32,33]. The 3-NTantibody-peptide(s) complex is allowed to form, and nonbinding peptides remaining in solution are removed by washing and analyzed by mass spectrometry as a supernatant fraction.…”

“…In an analogous manner, application of the proteolytic affinity-MS approach to ECP provided the identification of a single nitration site in the proteolytic peptide obtained by trypsin digestion, ECP( 23 CTIAMRAINNY(NO 2 )R 34 ) nitrated at Tyr 33 . The uncleaved Arg 28 residue suggested that the proteolytic resistance at this residue near the Tyr 33 residue might be due to the nitration, since in non-nitrated ECP the same arginine residue was cleaved by trypsin; the observation that proteolytic cleavage is affected by a nearby Tyr-nitration site has been made in several studies on protein Eosinophil proteins were isolated from four patients with hypereosinophilia and elevated eosinophils, using a previously described protocol [36].…”

“…In contrast, the direct identification of nitrations without affinity-extraction using HPLC-MS, such as in the auto-nitration site of eosinophilperoxidase (EPO) at Tyr 349 required highly purified protein and was only obtained after scrutinous HPLC separation of Figure 3. Identification of the tyrosine nitration site at Tyr −33 in eosinophil-derived neurotoxin by nano-ESI-FTICR-MS of the thermolysine peptide fragmnent, EDN (29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41)(42)(43). The modification denoted with X corresponds to carbamidomethylation at Cys-37.…”

“…It should be noted that quantitative determinations of the relative extent of nitrations using repetitive Edman sequencing can be carried out both with nitrated proteins in vitro, The locations of surface exposed tyrosine residues are indicated by colors as follows: yellow for Tyr 33 , red for Tyr 98 , and Tyr 123 and green for tyrosine hydroxyl groups. The structure modeling was performed using BallView 1.1.1 program based on the available X-ray crystal structure of EDN (PDB entry 1GQV) and with cellular proteins [36].…”

“…Recent work in our laboratory has focused on the development of combined affinity-mass spectrometry methods for the identification of biomolecular recognition structures, such as protein antigen-epitopes [29][30][31][32][33]. In this CI we describe a general approach for the structural identification of protein nitrations by a combination of proteolytic affinity extraction and ESI mass spectrometry ( Figure 1).…”