Characterization and Quantification of Poly(ADP-Ribose) in Cells and Tissues by Isotope Dilution Mass Spectrometry

Martello, Rita

Characterization and Quantification of Poly(ADP-Ribose) in Cells and Tissues by Isotope Dilution Mass Spectrometry

Files

Diss_Martello.pdf(10.65 MB)

Date

2013

Publication type

Dissertation

Abstract

Poly(ADP-ribose) (PAR) is a nucleic-acid like biopolymer of various chain lengths with a linear or branched structure which is synthesized by poly(ADP-ribose) polymerases (PARPs). PARPs are involved in various biological processes such as genomic maintenance, cell cycle progression and regulation of cell death. Moreover, PARP inhibitors are currently being evaluated in cancer therapy, either as radio- and chemosensitizers, or as monotherapeutic agents following the concept of synthetic lethality. A detailed analysis of the physiological role of PAR requires both an accurate knowledge of its structure and a reliable method for its quantification. This thesis reports the development of a sensitive, precise and accurate bioanalytical method, based on chromatography-coupled isotope-dilution tandem mass spectrometry, to characterize and quantify PAR with femtomol sensitivity and unequivocal chemical selectivity. To this end, PAR is extracted from cells, purified via solid-phase extraction, and hydrolyzed to specific monomeric units which are then subjected to mass spectrometric analysis. Non-radioactive 13C,15N-labeled PAR was synthesized and used as an authentic chemical standard to account for technical variability during sample preparation and mass spectrometric analysis. Using this method, it is now possible to quantify PAR in different human cancer cell lines, in primary human peripheral blood mononuclear cells (PBMCs), and in mouse tissue both under physiological conditions as well as upon induction of genotoxic stress.
This work also demonstrates several potential biological applications of this method. First, LC-MS/MS analysis revealed that DNA-damaging sulfur and nitrogen mustard derivatives robustly induce PARP activation in a dose-dependent manner in a human cancer cell line. Second, ex vivo pharmacodynamic studies were performed in order to define pharmacodynamic properties of clinically relevant PARP inhibitors in freshly isolated PBMCs. Third, poly(ADP-riboyl)ation was analyzed in a spectrum of different mouse tissues which revealed tissue-dependent differences in basal and DNA damage induced PAR levels. Finally a mouse model of systemic inflammation was characterized with regards to its poly(ADP-ribosyl)ation status.
In conclusion, this study reports a novel mass spectrometric method for quantifying cellular PAR levels that will be instrumental for in-depth investigations on the metabolism of poly(ADP-ribosyl)ation.

Summary in another language

Poly(ADP-Ribose) (PAR) stellt ein Biopolymer mit variabler Kettenlänge dar. Es besitzt nukleinsäure-ähnliche Eigenschaften und weist eine lineare oder verzweigte Struktur auf. PAR wird durch Poly(ADP-Ribose) Polymerasen (PARP) synthetisiert. Dies sind Enzyme, die bei der Regulation etlicher biologischer Prozesse beteiligt sind, wie z.B. der Erhaltung der genomischen Stabilität, der Zellzyklus-Regulation, und bei Mechanismen der Zelltod-Induktion. PARP Inhibitoren werden zur Zeit als mögliche Therapeutika in der Krebstherapie getestet, zum einen in Kombination mit Radio- oder Chemotherapie, zum anderen als monotherapeutische Agenzien, dem Konzept der synthetischen Lethalität folgend. Eine genaue Analyse der Funktion der Poly(ADP-Ribosyl)ierung in Physiologie und Pathophysiologie bedarf einer detailierten strukturellen Charakterisierung und einer verlässlichen Methode zur zellulären Quantifizierung der PAR.
Diese Dissertation beschreibt die Entwicklung und Validierung einer sensitiven, präzisen und exakten bioanalytischen Methode basierend auf HPLC-gekoppelter „Isotope-dilution“ Massenspektrometrie (LC-MS/MS), um PAR mit höchst-möglicher Sensitivität und chemischer Selektivität zu charakterisieren und zu quantifizieren. Hierzu wurde PAR aus Zellen extrahiert, mittels Festphasenextraktion aufgereinigt und zu charakteristischen monomeren Einheiten hydrolysiert, welche massenspektrometrisch analysiert wurden. Nicht-radioaktives 13C,15N-markeriertes PAR wurde synthetisiert und als authentischer, chemischer Standard eingesetz, um die technische Varianz in der Probenvorbereitung und massenspektrometrischen Messung zu minimieren. Mit dieser Methode wurde PAR in verschiedenen Krebszelllinien, in humanen peripheren mononukleären Blutzellen (PBMC), sowie in Mausgeweben unter physiologischen Bedingungen als auch nach Induktion von genotoxischem Stress gemessen.
Diese Arbeit zeigt weiterhin einige mögliche Anwendungen dieser Methode auf: Erstens, es konnte mittels LC-MS/MS Analyse gezeigt werden, dass DNA-schädigende Schwefel- und Stickstoff-Lost Derivate die zelluläre PARP Aktivität dosis-abhängig stimulieren. Zweitens, es wurden ex-vivo Studien an frisch-isolierten PBMC durchgeführt, um die pharmakodynamischen Eigenschaften klinisch relevanter PARP Inhibitoren zu bestimmen. Drittens, die Analyse basaler und stress-induzierter PAR Level in verschiedenen Mausgeweben zeigte einige gewebe-abhängige Unterschiede der Poly(ADP-Ribosyl)ierung auf. Weiterhin wurde ein Mausmodel einer systemischen Entzündungsreaktion in Hinblick auf dessen Poly(ADP-Ribosyl)ierungsstatus analysiert. Zusammenfassend beschreibt die vorliegende Arbeit die Entwicklung, Validierung und Anwendung einer neuen massenspektrometrischen Methode zur Quantifizierung zellulärer Poly(ADP-Ribose), welche als Grundlage für detailierte Untersuchungen des Poly(ADP-Ribose)-Metabolismus genutzt werden kann.

Subject (DDC)

570 Biosciences, Biology

Keywords

Mass Spectrometry,Poly(ADP-Ribose),DNA damage

Cite This

ISO 690

MARTELLO, Rita, 2013. Characterization and Quantification of Poly(ADP-Ribose) in Cells and Tissues by Isotope Dilution Mass Spectrometry [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Martello2013Chara-23025,
  year={2013},
  title={Characterization and Quantification of Poly(ADP-Ribose) in Cells and Tissues by Isotope Dilution Mass Spectrometry},
  author={Martello, Rita},
  address={Konstanz},
  school={Universität Konstanz}
}

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Examination date of dissertation

March 15, 2013