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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Titel in einer weiteren Sprache
Analytische Entwicklung und biochemische Anwendung der Kombination von Massenspektrometrie und Immunoaffinitätsanalytik zur Strukturaufklärung der Nitrierung von Proteinen
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Zusammenfassung

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.

Zusammenfassung in einer weiteren Sprache

Die Nitrierung von Proteinen ist eine posttranslationale Modifikation, die in jüngster Zeit unter physiologischen Bedingungen nachgewiesen wurde und Interesse gefunden hat, und die unter pathophysiologischen Bedingungen als oxidative Stress Modifizierung verstärkt auftritt. Zur Ermittlung von molekularen Beziehungen zwischen Protein-Nitrierung und pathogenen Mechanismen von Erkrankungen beim Menschen sowie in Tiermodellen ist die Identifizierung von infizierten Targetproteinen und die Aufklärung der modifizierten Strukturen von entscheidender Bedeutung. In der vorliegenden Dissertation ermöglichte die Entwicklung von neuen analytischen und immunologischen Methoden die Identifizierung und quantitative Bestimmung von Tyrosin-nitrierten Proteinen. Spezifische, Tyrosin-nitrierte Proteinstrukturen sind als mögliche oxidative Biomarker von Bedeutung. Die Entwicklung und Anwendung von neuen immun-analytischen und massenspektrometrischen Methoden zu entsprechenden Untersuchungen in vitro und in vivo waren zentrale Zielsetzungen der Dissertation. Im ersten Teil der Dissertation wurde die Tyrosin-Nitrierung der Prostacyclin-Synthase (PCS) aus Rinderaorta- Mikrosomen nach Oxidation durch Peroxynitrit untersucht. Durch eine Kombination von proteolytischem Abbau, HPLC und Nano- ESI-FTICR-Massenspektrometrie wurde eine spezifische Nitrierung von Tyr-430 nachgewiesen. Das identifizierte nitrierte Tetrapeptid PCS (427-430) wurde durch eine ungewöhnliche Spaltung mit Thermolysin erhalten, was Hinweise auf eine geringe Zugänglichkeit der Modifizierungsstelle lieferte. Der Vergleich mit der Röntgenkristallstruktur der PCS bestätigt, dass der modifizierte Tyr-430 in dem eng gefalteten, wenig zugänglichen katalytischen Zentrum der PCS liegt, und somit die hohe Resistenz dieser Domäne gegenüber Proteasen erklärt. In einem zweiten Teil der Dissertation wurde die physiologische Tyr-Nitrierung in menschlichen Eosinophil-Proteinen untersucht. In den Proteinen Eosinophil-Peroxidase (EPO), Eosinophil-Kationisches Protein (ECP) und Eosinophil-derived neurotoxin (EDN) konnten Tyr-nitrierte Strukturen erstmals unter physiologischen Bedingungen nachgewiesen werden. Erste Hinweise auf Nitrierung der Eosinophil- Proteine wurden aus Westernblot-Daten mit anti-Nitro-Tyr Antikörpern erhalten. Für die Eosinophil-Peroxidase wurde eine spezifische Nitrierung von Tyr-349 durch HPLC-Tandem-MS mit strukturspezifischer UV-Detektion nachgewiesen. Dabei zeigten Molekülmodellstudien, dass der Tyr-349 Rest in einem flexiblen Loop-Bereich liegt, dessen Flexibilität durch sterische- und Ladungs Effekte aufgrund der Nitrierung verlorengeht; die ohne Modifizierung vorliegende Wechselwirkung mit dem Tyr-555 Rest geht dadurch verloren und die Niro-Tyr-349 wird an der Proteinoberfläche exponiert. Die meist sehr geringe Nitrierungsrate von Proteinen erschwert die Identifizierung von Tyr- Nitrierungen. In der vorliegenden Arbeit wurde eine neue, hochempfindliche und spezifische Methode zur Identifizierung von Tyr-Nitrierungen aus biologischem Material durch Kombination von proteolytischer Affinitätsextraktion und Massenspektrometrie entwickelt (PROFINEX-MS). Die Anwendung dieser Methode ermöglichte erstmals die Aufklärung der Nitrierungen von Tyr-33 des ECP Proteins, sowie des analogen Tyr-33 Rests des EDN Proteins aus menschlichen Eosinphilen. Die Strukturen der Modifizierungen wurden zusätzlich durch Tandem-MS Analyse der Fragmentpeptide gesichert. Mit Hilfe der Affinitäts-MS Methode sowie Edman-Sequenzanalyse wurden erstmals auch quantitative Abschätzungen der Nitrierungsraten erhalten, die für die Eosinophilproteine ECP und EDN bei ca.10-15 % liegen. Konformationsuntersuchungen der Tyr- nitrierten Peptide mittels CD-Spektroskopie zeigen hohe Flexibilität und ungeordnete Struktur des Nitrotyrosinrests. In einem weiteren Teil der Arbeit wurden Nitrotyrosin-haltige Modellpeptide durch Festphasensynthese synthetisiert, massenspektrometrisch und immunanalytisch charakterisiert. Die Darstellung der nitrierten Peptide gelang mit der Fmoc- Methode in hoher Ausbeute und Reinheit. Vergleichende Untersuchungen mittels ESI-MS und MALDI-MS zeigten, dass NitroTyr-Peptide unter MALDI-Bedingungen mit Stickstoff-UV-Laser eine charakteristische photochemische Fragmentierung der Nitro-Tyr-Gruppe eingehen; dagegen wurden mit der IR- MALDI-MS stabile Molekülionen ohne Zersetzung erhalten. In Ergänzung und Erweiterung der standardmäßigen immunoanalytischen Methoden, Dotblot und ELISA, wurde die Affinitäts-Massenspektrometri im letzten Teil der Arbeit zur molekularen Charakterisierung der Spezifität von Anti-Nitrotyrosin Antikörpern angewendet. Die Ergebnisse zeigen unterschiedliche Erkennungsspezifitäten von kommerziell erhältlichen Antikörpern, die zur Entwicklung von spezifischem Biomarkern der Tyr-Nitrierung genutzt werden können. Mit Hilfe der Affinitäts-MS konnte ein spezifisches Epitopmotiv der Erkennung von Nitrotyrosin- Peptidsubstraten identifiziert werden.

Fachgebiet (DDC)
540 Chemie
Schlagwörter
Nitrierung von Proteinen, Nitro-tyrosine, Massenspektrometrie, Immunoaffinitätsanalytik, Protein nitration, nitro-tyrosine, mass spectrometry, immunoaffinity-MS
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Zitieren
ISO 690PETRE, Brînduşa-Alina, 2008. Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration [Dissertation]. Konstanz: University of Konstanz
BibTex
@phdthesis{Petre2008Analy-9703,
  year={2008},
  title={Analytical Development and Biochemical Application of Mass Spectrometry in Combination with Immunoaffinity Methods for Identification and Structural Characterisation of Protein Nitration},
  author={Petre, Brînduşa-Alina},
  address={Konstanz},
  school={Universität Konstanz}
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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. 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May 28, 2008
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