Project description

Scientific context and motivation. The nitration of tyrosine residues in protein represents an important post-translational modification during development, oxidative stress, and biological aging; however it is difficult to be detected specifically. Nitration is a covalent modification in proteins resulting from the addition of a nitro group onto one of the two equivalent aromatic carbons adjacent to the hydroxyl group of a tyrosine residue. This modification leads to changes in protein structure and function. Only a selective number of proteins are modified by nitration in vivo, and the selectivity may be influenced by a combination of several factors such as (1), the proteins are in close proximity to the site of generation of nitrating agents; (2), the chemical selectivity of the nitrating reagent; (3), the relative abundance of the target proteins; (4), the proteins contain tyrosine residues in a specific primary sequence or in a specific environment and (5), the repair of nitrated proteins by a putative enzyme called denitrase [1].

In order to identify protein nitrations in biomedical samples and to assess their relevance for health effects, analytical methods of high sensitivity and molecular specificity are required. Several methods have been used for the detection and identification of 3-NT including high-performance liquid chromatography (HPLC) in combination with UV/VIS detection, fluorescence detection after derivatisation[2], gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS)[3-5], and various immunochemical techniques[6, 7]. A major part of studies on tyrosine nitration in tissues and biological fluids has been carried out by antibody- based methods, such as immunohistochemistry, immunoprecipitation, Western blot, and ELISA. Several commercially available monoclonal and polyclonal antibodies have been employed in different studies, e.g. to demonstrate increased levels of 3-nitrotyrosine in lung tissue from patients with cystic fibrosis, chronic hepatitis and Parkinson’s and Alzheimer’s disease [8, 9]. The present results indicate that immunoassays critically depend on the – frequently uncharacterized – specificities of antibodies. Moreover, these methods only provide overall information of nitration, while the specific identification of nitration sites requires molecular characterization methods such as mass spectrometry.

Mass spectrometry using electrospray ionization (ESI-MS) provides unambiguous identification of nitrotyrosine-modified peptides by the increase in molecular mass by 45 amu due to the nitro group. In contrast, UV-MALDI- MS using a standard nitrogen laser (337 nm) leads to photochemical decomposition of nitrotyrosyl residues and other oxidative modifications, and hence present problems for their identification in biological material. The newly introduced infrared-MALDI ionisation, have been developed during my PhD work as powerful approaches for unequivocal and sensitive identification of nitrated tyrosine containing peptides, providing more stable ions than in case of using UV-MALDI-ionisation [10].

In recent years a combination of 1D,-2D-electrophoresis, western blotting, and mass spectrometry has been applied in proteomics studies of nitrated proteins from biological material. Gokulrangan et al. used 2D gel electrophoresis and Western blot for isolation and detection of 3NT-containing proteins, which were subsequently identified by mass spectrometry [11] however the specific 3- nitrotyrosine modification sites were not determined. The failure of many 2-DE approaches to characterize such nitrated proteins is likely due to multiple causes such as (i) the low abundance of some of the NT-containing proteins, (ii) 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 (iii) the recovery of NT-containing peptides from the gels and /or HPLC columns during subsequent liquid chromatography-MS analysis. In many previous studies only the proteins and not the Tyr-nitration structures were identified by proteome analysis, which renders the identification of nitrations critically dependent on the specificity of NT-antibodies. The major problems for identification (i), the low degree of nitrations, and (ii), the low stability of modified structures, have been overcome by the recent development of affinity-MS methods, which will be employed as main tools in this project. Affinity – mass spectrometry methods, in combination with proteolytic digestion, have been previously developed and employed (i), for the identification of antigen – epitopes (epitope – excision and – extraction-MS)[12] and (ii), in an affinity- proteomics approach which enables direct protein identification from biological material with unprecedented selectivity [13]. The major goal of this project is to apply and improved a proteolytic-affinity mass spectrometric approach for the identification and structural characterization of tyrosine nitration in complex biological samples.

The oxidative processes such as (i) tyrosine nitration will increase α-Synuclein fibril formation [14] and (ii) oxidation of methionine 35 reduces the neurotoxicity and delayed Aβ fibril formation [15], opened a new door in our research outline for studying the aggregation phenomena of several proteins. Understanding pathophysiological protein aggregation is a key prerequisite for the development of drugs capable of neutralizing or disaggregating aggregates or inhibiting aggregate formation. At present, we still lack the detailed characterization of aggregating protein intermediates and only methods combine with mass spectrometry can determine their structural mass. In this project, we will develop mass spectrometry-based approaches for the characterization of molecular structure and reaction pathways of aggregates of neurodegenerative related proteins such as Aβ–amyloid peptides, α-synuclein and eosinophil cationic protein.

References:

[1] S. M. Stevens, Jr., K. Prokai-Tatrai, L. Prokai, Mol Cell Proteomics 2008, 7, 2442.