Scientific context and motivation<\/span>. 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 <\/span>in vivo<\/span>, 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 <\/span>[1]<\/span>. <\/span><\/p>\n

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<\/span>[2]<\/span>, gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS)<\/span>[3-5]<\/span>, and various immunochemical techniques<\/span>[6, 7]<\/span>. 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 <\/span>cystic fibrosis, chronic hepatitis and Parkinson\u2019s and Alzheimer\u2019s disease <\/span>[8, 9]<\/span>. The present results indicate that immunoassays critically depend on the <\/span>\u2013 <\/span>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. <\/span><\/p>\n

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 <\/span>[10]<\/span>. <\/span><\/p>\n<\/div>\n<\/div>\n<\/div>\n