Global Cardiac Phosphoproteome Analysis In Nitric Oxide-Induced Heart Failure Inaugural-Dissertation to obtain the scientific degree Dr. rer. nat. of the Department of Mathematics and Natural Sciences of the Heinrich-Heine-University Düsseldorf submitted by Annamária Simon from Keszthely (Hungary) Düsseldorf, June 2009 From the Institute of Cardiovascular Physiology of the Heinrich-Heine-University Düsseldorf Printed with the authorization of the Department of Mathematics and Natural Sciences of the Heinrich-Heine-University Düsseldorf First Supervisor: Prof. MD Jürgen Schrader Second Supervisor: Prof. Dr. Rainer Weinkauf Day of the oral examination: 16. July 2009 This work is dedicated to my family ACKNOWLEDGEMETNS ACKNOWLEDGEMENTS First and foremost I would like to thank Prof. Dr. med. Jürgen Schrader for giving me the opportunity to work at his laboratory. I am deeply grateful for his excellent supervision at every stage of this work. Furthermore, I would like to thank Prof. Dr. Rainer Weinkauf for the fast preparation of the expert opinion. I would also like to thank the Graduate College 1089 “Protein interactions and modifications in the heart“ for the interesting courses and for the financial support. Additionally I would like to thank Prof. Dr. Albert Sickmann for giving me some practical tips and tricks in proteomics. I am grateful to have the possibility of performing some test measurements on LTQ Orbitrap XL at Thermo Scientific in Bremen. Furthermore, I would like to thank Dr. Michael Reinartz and Dr. Urlich Flögel for performing the heart perfusion experiments, additional thanks to Dr. Michael Reinartz also for mentoring me during my PhD thesis and for the critical reading of this manuscript. I would also like to thank Dr. Christoph Jacobi for helping me in computer problems and for programming MSanalysis. Hereby I would like to thank Dr. Peter Schneider for taking care of the MASCOT server cluster and introducing me into the basics of intranet to reach analyzed results and used databases. Additionally, I would like to thank Dr. Peter Mortensen for providing dimethyl labeling compatible version of MSQuant and Dr. Bas van Breukelen for some bioinformatics programs enabling easier and quantitative analysis of measured data. I am especially grateful for Jan Heye Buß for helping me in the MATLAB program based proteomic data management. Furthermore, I would like to thank all my colleagues and PhD students from the Institute of Cardiovascular Physiology and from the Graduate School 1089 for fruitful scientifical discussions and for a nice atmosphere at the laboratory. At last but not at least I thank my family and all my friends, who supported and motivated me during the last years. TABLE OF CONTENTS TABLE OF CONTENTS ABSTRACT.................................................................................................8 ABSTRAKT (IN GERMAN)...........................................................................9 ABBREVIATIONS.......................................................................................10 I.INTRODUCTION ...............................................................................17 PHYSICAL AND CHEMICAL PROPERTIES OF NO ............................................................17 NITRIC OXIDE AS REGULATOR MOLECULE IN THE HEART .........................................18 NO SYNTHESIS – THE NOSS .........................................................................................................................................18 NO DEPENDENT SIGNALING PATHWAYS ......................................................................................................................20 HEART FAILURE AND NO ...............................................................................................................................................22 HEART FAILURE MODEL (INOS+/MYO-/-) MICE ...........................................................................................................23 MASS SPECTROMETRY IN PROTEOMICS ...........................................................................25 PRINCIPLE ........................................................................................................................................................................25 ION SOURCES ..................................................................................................................................................................25 MASS ANALYZER ..............................................................................................................................................................26 ANALYSIS OF PROTEIN PHOSPHORYLATION USING MASS SPECTROMETRY .................................................................28 OVERVIEW OF MS BASED QUANTIFICATION STRATEGIES ............................................................................................29 II. AIM OF THE STUDY ..........................................................................32 III. MATERIALS AND METHODS ..........................................................33 USED MATERIALS ....................................................................................................................33 CHEMICALS AND MATERIALS .........................................................................................................................................33 MS STANDARDS ...............................................................................................................................................................34 ANIMALS ..........................................................................................................................................................................34 PRIMARY AND SECONDARY ANTIBODIES .......................................................................................................................34 LABORATORY INSTRUMENTS ..........................................................................................................................................35 SOFTWARE .......................................................................................................................................................................36 LANGENDORFF-PERFUSION OF ISOLATED MOUSE HEARTS ......................................37 BIOANALYTICAL METHODS .................................................................................................37 PROTEIN ASSAY USING BICINCHONINIC ACID (BCA) .................................................................................................37 TWO DIMENSIONAL GEL ELECTROPHORESIS (2-D PAGE) .........................................................................................38 ISOELECTRICAL FOCUSING (1. DIMENSION) ..........................................................................................................38 GRADIENT SDS-PAGE (2. DIMENSION) ...............................................................................................................39 PROQ DIAMOND PHOSPHOPROTEIN STAIN ................................................................................................................40 MS COMPATIBLE SILVER STAIN ........................................................................................................................................40 COLLOIDAL COOMASSIE STAIN .....................................................................................................................................40 WESTERN BLOT ...............................................................................................................................................................41 IN GEL DIGESTION WITH TRYPSIN ..................................................................................................................................42 PREPARATION OF HEART SAMPLES FOR COMPARATIVE PHOSPHOPROTEOME ANALYSIS USING STABLE ISOTOPE DIMETHYL LABELING .........................................................................................................................................................................42 PROTEIN EXTRACTION AND FRACTIONATION FOR IN SOLUTION DIGEST ..........................................................42 INTERNAL STANDARD – A REFERENCE FOR RELATIVE QUANTITATIVE ANALYSIS ..................................................42 REDUCTION AND ALKYLATION OF CYSTEINE RESIDUES ........................................................................................43 METHANOL-CHLOROFORM PRECIPITATION OF PROTEINS ....................................................................................43 5 TABLE OF CONTENTS ETHANOL PRECIPITATION ........................................................................................................................................43 PROTEIN DIGESTION .................................................................................................................................................43 STABLE ISOTOPE DIMETHYL LABELING .....................................................................................................................43 SAMPLE CLEAN UP USING SOLID PHASE EXTRACTION ...........................................................................................44 PHOSPHOPEPTIDE ENRICHMENT USING TITANIUM DIOXIDE MICRO PARTICLES FILLED TOPTIP .........................44 PHOSPHOPEPTIDE ENRICHMENT USING CALCIUM PHOSPHATE PRECIPITATION ..................................................45 SAMPLE FRACTIONATION USING SCX FILLED TOPTIP .........................................................................................45