Problems in using analytical methods for identification of protein nitration .18

The nitration of tyrosine residues in protein represents an important post-translational modification during development, oxidation stress, and biological aging; however it is difficult to be detected. Several years ago Halliwell suggested: “… an under-addressed problem is the reability of assays used to detect and measure 3-nitrtyrosine in tissues and body fluids: immunostaining results vary between laboratories and simple HPLC methods are susceptible to artifacts. Exposure of biological material to low pH (e.g., during acidic hydrolysis to liberate nitro-tyrosine from proteins) or to H2O2 might cause artifactual generation of nitro-tyrosine from NO2^–in the samples. This may be the origin of some of the very large values from tissue nitro-tyrosine levels quoted in the literature” [112]. The ability of methods to specifically measure nitrated substrates; in complex mixture is dependent on a wide range of parameters, including the nature of the nitrating agents for in vitro experiments, the nature of the antibody, sample types, amount, other components present and time.

One of the major problems is the site-selectivity of tyrosine nitration in proteins.

Schöneich et al. showed for creatine kinase, that the selectivity of in vivo nitration does not correspond to the product selectivity of in vitro studies, where exclusively Tyr⁸² was nitrated when creatine kinase was exposed to peroxynitrite (s. Table 2).

These studies demonstrated that the in vitro exposure of an isolated protein to peroxynitrite may not always be a good model to mimic protein nitration in vivo; and is probably depending on the corresponding concentration of RNS. Albumin was modified chemically with tetranitromethane (TNM), and several 3-nitro-tyrosine residues were identified by LC MS/MS analysis of tryptic BSA peptide mixture [108].

Other unspecific tyrosine sites were reported to be nitrated, by treatment of BSA with peroxynitrite [106, 107]. Clearly the nitrating agent and reaction conditions can influence the structure and the extent of chemical modification; however the factors determining the selectivity of tyrosine nitration remain unclear.

The specificity and sensitivity of the different methods in the identification of nitrated proteins and/or nitrated tyrosine sites varies greatly and false positive or negative detection of nitro-tyrosine in proteins may result. Most protein nitrations have been identified by antibodies directed against 3-nitro-tyrosine, and only little information on the properties of these antibodies has been published. Franze et al. characterized and compared three monoclonal and three polyclonal 3-NT antibodies with respect to their cross-reactivities and affinities for free 3-nitrotyrosine, synthetic nitrated peptides and nitrated proteins. They observed that a mouse monoclonal antibodies exhibited the highest affinity for free 3-NT, while a polyclonal antibodies exhibited the highest affinities for nitrated proteins [113]. In order to characterize possible false negative or positive responses obtained by using anti 3-NT antibodies, two types of negative control experiments were reported in the literature (i) blockade of 3-NT antibody with pure nitro-tyrosine free amino acid and (ii) reduction to nitro-tyrosine to amino-tyrosine in proteins. Both of these experiments present problems that will be discussed in the next Chapter 2.1.

Both MALDI and ESI mass spectrometry allow the assignment of protein nitration and the nitrated structures in proteins. The fragmentation of nitrated peptides observed by using UV-MALDI laser radiation however, reduces the abundance of the signal for nitrated peptide, resulting in failure to observe such signals in complex peptide mixtures. Therefore, IR-MALDI-FTICR-MS was used first for the characterization of 3-nitro-tyrosine containing peptides and was found as a successfully application for proteome studies of Tyrosine nitration. By using ESI tandem MS/MS for identification of nitro-tyrosine-containing peptides, the characteristic 3-NT immonium ion at m/z 181 does appear to be generally present in the mass spectra of pure standards, but typically at a relatively low intensity, so it is an unreliable indicator of the presence of nitrated tyrosine in the complex mass chromatograms of protein digests.

To rationalize any physiological changes with such modifications, the actual protein nitrated structures must be identified by proteomics methods. While several studies have used proteomics to screen for 3-nitrotyrosine-containing proteins in vivo, most

The failure of many 2-DE approaches to characterize such nitrated proteins is likely due to multiple causes such as (i) the low steady-state level of 3-NT on specific proteins, (ii) the low abundance of some of the 3-NT-containing proteins, (iii) the solubility, size, hydrophobicity and/or extreme pI values of proteins, which may compromise the isoelectric-focusing in the first step of the 2-DE separation, and (iv) the recovery of 3-NT-containing peptides from the gels and /or HPLC columns during subsequent liquid chromatography-MS analysis.

In conclusion, nitro-tyrosine modification presents particular challenges because of the low levels present in vivo and the potential for artifact formation, therefore require methods which provide a molecular chemical identification are required.

1.5 Scientific goals of the dissertation

The detailed characterisation of nitro-tyrosine containing proteins as well as other post-translational modified proteins is required to fully understand protein function and regulatory events in the cell and organisms. Oxidative modification of proteins may cause substantial biochemical changes as well as pathophysiological consequences, both by chemical reactions and specific enzymatic pathways;

however, the identification of corresponding fine-structure modifications is often tedious and requires methods of high sensitivity and molecular specificity. Nitration of Tyrosine residues has been associated to pathophysiological effects in proteins related to neurodegeneration such as in Alzheimer's disease, Parkinson’s disease, atherosclerosis, and broncho-alveolar diseases. While immuno-analytical methods suffer from low detection specificity of antibodies, mass spectrometric methods for identification of Tyrosine nitration are hampered by low stabilities and levels of modification, and by possible changes of structure and proteolytic degradation. In the present work, new mass spectrometric methods have been developed as powerful approaches for unequivocal and sensitive identification of tyrosine-nitrations in

The major objectives of the dissertation are summarized as follows:

1. Development of a new affinity - mass spectrometric approach for specific identification of nitro- tyrosine sites in proteins.

2. Mass spectrometric applications for the identification of tyrosine nitration sites, (i) in prostacyclin synthase upon peroxynitrite treatment at bovine aortic microsomes, and (ii) of specific endogenous physiological nitration in human eosinophil proteins.

3. Structural modelling investigations of identified 3-nitro-tyrosine residues in proteins, for the elucidation of site selectivity of this modification.

4. Synthesis of nitrated tyrosine-containing targets peptides for developing different analytical strategies such as ESI and MALDI mass spectrometry, Dot blot, ELISA and immuno-affinity – MS methodologies.

5. Evaluation of molecular recognition properties and selectivity of anti 3-nitro-tyrosine antibodies with 3-nitro-3-nitro-tyrosine peptide substrates.