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Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration

Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration

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PETRE, Brînduşa-Alina, 2008. Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration

@phdthesis{Petre2008Analy-9703, title={Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration}, year={2008}, author={Petre, Brînduşa-Alina}, address={Konstanz}, school={Universität Konstanz} }

<rdf:RDF xmlns:rdf="http://www.w3.org/1999/02/22-rdf-syntax-ns#" xmlns:bibo="http://purl.org/ontology/bibo/" xmlns:dc="http://purl.org/dc/elements/1.1/" xmlns:dcterms="http://purl.org/dc/terms/" xmlns:xsd="http://www.w3.org/2001/XMLSchema#" > <rdf:Description rdf:about="https://kops.uni-konstanz.de/rdf/resource/123456789/9703"> <dcterms:abstract xml:lang="eng">Protein nitration is a posttranslational modification which may occur under physiological conditions, but may be enhanced under various pathological conditions associated with oxidative stress. For the molecular correlation of protein nitration with pathogenic mechanisms of human diseases and with animal models of diseases it is essential to identify the protein targets of nitration and the specific modification sites. In this work the development of analytical and immunological methodologies allowed the identification and the quantitative estimation of tyrosine nitrated proteins. Specific nitro-Tyr structures in given proteins may be of importance as oxidative biomarkers. The development of new immunoanalytical methods and their mass spectrometric applications in in vivo and in vitro studies were the major goals of the present dissertation. In the first part of the thesis, the Tyr-nitration in Prostacyclin synthase (PCS) upon treatment of bovine aortic microsomes by peroxynitrite was identified at Tyr-430 residue using a combination of proteolytic fragmentation, HPLC and nano-ESI-FTICR MS. The identified nitrated tetra peptide (PCS 427-430) resulted from an unpredicted thermolysine cleavage. According to the X-ray structure of PCS the Tyr-430 and heme bound Cys-441 form a loop around the heme and this structure is embedded in a tightly folded conformation. This structure explains the resistance to proteases, and the difficulties for obtaining reproducible identification of nitrated PCS by Western blots, caused by protein refolding on the blotting membrane. A second part of the dissertation was focused on the identification of physiological Tyr nitration in human eosinophil proteins isolated from patients with hypereosinophilia, elevate eosinophil number in blood. The close relationship between eosinophilia and nitro-Tyr formation suggested that the eosinophil peroxidase (EPO) itself is an important factor in promoting protein nitration. First evidence for nitration of eosinophil proteins was obtained from Western blot using an anti 3-nitro-Tyr antibody. The selective nitration at Tyr349 in EPO was identified using HPLC-ESI-MS with specific UV detection for nitro-Tyr. A molecular modelling study showed that Tyr349 is present in a highly flexible loop region, in a weak, parallel stacking interaction with Tyr555. Upon nitration at Tyr349, the flexibility of this loop is lost due to steric hindrance and the net electronegativity of nitro-Tyr residue likely prevents the stacking interactions with Tyr555, forcing the nitro group to be permanently surface exposed. A new method was developed to specifically identify Tyr nitration by a proteolytic affinity extraction (PROFINEX) approach combined with high resolution mass spectrometry. This method, analog to the highly efficient epitope-identification-MS methods was found to be sensitive and specific to identify low level of Tyr nitrations in biological samples. Using this approach specific nitro-Tyr was identified at Tyr33 for eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). By mean of tandem MS/MS analysis additional proof for the modified structure at Tyr33 was obtained. Furthermore, a combination of the proteolytic affinity peptide extraction and Edman sequencing provided quantitative information of the endogenous nitration in eosinophil proteins. Conformation studies of nitrated peptides by circular dichroism and molecular modeling studies indicated that Tyr33 is located on a flexible, random coil structure which exposes the Tyr residue to the protein surface. In a further part of this thesis, Tyr and nitro-Tyr peptides were synthesized by solid-phase peptide synthesis accordingly to Fmoc strategy. The synthetic peptides were characterized by mass spectrometry and used for immunoanalytical studies. Using UV-MALDI-MS, nitrated peptides were found to undergo photochemical fragmentation at nitro-phenyl group, which may hamper the unequivocal identification of Tyr-nitration in proteins. The application of IR-MALDI-FTICR was shown as a useful method for analysis of nitro-Tyr containing peptides which undergo photochemical fragmentation under UV-MALDI conditions but were stable under IR laser radiation. In complementing the conventional immunoanalytical techniques such as Dot blot and ELISA, affinity- MS was shown in the last part of the thesis as a powerful tool for characterising recognition specificities of different antibodies. The comparison of these methods provided results to reveal differences in binding affinities and specificities by nitro-Tyr antibodies, showing that antibody binding is influenced by the peptide structure adjacent to the nitro-Tyr site. The capability of anti nitro-Tyr antibodies to discriminate between nitro-Tyr in different environments in proteins may be useful for producing antibodies to specific motifs containing Tyr residues, and for the development of highly specific biomarkers.</dcterms:abstract> <dc:date rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T18:13:49Z</dc:date> <dcterms:rights rdf:resource="http://nbn-resolving.org/urn:nbn:de:bsz:352-20140905103416863-3868037-7"/> <dcterms:available rdf:datatype="http://www.w3.org/2001/XMLSchema#dateTime">2011-03-24T18:13:49Z</dcterms:available> <dc:contributor>Petre, Brînduşa-Alina</dc:contributor> <dcterms:title>Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration</dcterms:title> <dcterms:issued>2008</dcterms:issued> <bibo:uri rdf:resource="http://kops.uni-konstanz.de/handle/123456789/9703"/> <dc:rights>deposit-license</dc:rights> <dc:creator>Petre, Brînduşa-Alina</dc:creator> <dc:format>application/pdf</dc:format> <dc:language>eng</dc:language> <dcterms:alternative>Analytische Entwicklung und biochemische Anwendung der Kombination von Massenspektrometrie und Immunoaffinitätsanalytik zur Strukturaufklärung der Nitrierung von Proteinen</dcterms:alternative> </rdf:Description> </rdf:RDF>

Dateiabrufe seit 01.10.2014 (Informationen über die Zugriffsstatistik)

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