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.

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

“…As pointed out in recent studies [33,46], the characterization of affinities and binding specificities of monoclonal 3-NT-specific antibodies are of crucial importance and should be ascertained using synthetic tyrosine-nitrated peptides; high antibody affinities were found for nitrotyrosine peptides containing basic (Lys, Arg) amino acid residues in the vicinity of the nitrated tyrosine ( Figure 5). Corresponding tyrosine-nitrated peptides may also find important application as biomarkers of oxidative modifications.…”

“…Determination of antibody/ protein association kinetics was performed by extracting the data from the sensor signals using the monomolecular growth model [47]. Determinations of dissociation constants was performed by plotting the pseudo first-order kinetic constants for two independent channels verss concentration, and applying linear regression for K D =k off k on -1 for nitration sites, comprising cationic amino acids in the vicinity of nitrated tyrosine residues (Table 2) [46]. The possible role of sequence and structural environment for nitration has been discussed by several authors [11,23,33,36,40,[46][47][48].…”

“…Determinations of dissociation constants was performed by plotting the pseudo first-order kinetic constants for two independent channels verss concentration, and applying linear regression for K D =k off k on -1 for nitration sites, comprising cationic amino acids in the vicinity of nitrated tyrosine residues (Table 2) [46]. The possible role of sequence and structural environment for nitration has been discussed by several authors [11,23,33,36,40,[46][47][48]. Ischiropoulos et al [47] have pointed out the importance of the protein structure for regulation of nitration sites, which is consistent with our results on surface accessibilities in the nitration of eosinophil proteins (Figure 4).…”

“…The online affinity-MS approach utilized a surface-acoustic wave (SAW) biosensor [51], with the anti-3NT antibody immobilized on the biosensor chip surface via a self-assembled monolayer. Following association of peptides, dissociation was performed using an acidic glycine buffer into the interface for ESI-MS analysis [46]. Biosensor association curves and subsequent dissociation of affinity-bound ECP peptides from a mixture of nitrated and non-nitrated peptides are shown in Figure 5a.…”

Affinity-Mass Spectrometry Approaches for Elucidating Structures and Interactions of Protein–Ligand Complexes

“…3.2 ) the use of anti-3-nitro-tyrosine-specifi c antibodies has been essential for mass spectrometric identifi cation of tyrosine nitrations using proteolytic affi nity-MS. Poor information regarding the specifi cities of anti-3-nitrotyrosine antibodies has been reported before [ 4 ]. In a previous publication we described a molecular study of the recognition specifi cities and affi nities of two commercially available, monoclonal anti-nitrotyrosine antibodies by dot-blot, ELISA, and affi nity-MS, using different 3-nitrotyrosine-containing peptides [ 36 ].…”