Global cardiac phosphoproteome analysis in nitric oxide-induced heart [Elektronische Ressource] / submitted by Annamária Simon

Jan Heye Buß

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• Gesundheit

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2009
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 9 Mo

Extrait

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 2009From 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 familyACKNOWLEDGEMETNS
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
SYNTHESIS – THE NOSS 18.........................................................................................................................................NO
DEPENDENT SIGNALING PATHWAYS 20......................................................................................................................NO
EART FAILURE AND NO 22................H
EART FAILURE MODEL (INOS+/MYO-/-) MICE 23...........................................................................................................H
MASS SPECTROMETRY IN PROTEOMICS ...........................................................................25
RINCIPLE .........................................25P
ON SOURCES ...................................25I
ASS ANALYZER ...............................26M
NALYSIS OF PROTEIN PHOSPHORYLATION USING MASS SPECTROMETRY 28.................................................................A
VERVIEW OF MS BASED QUANTIFICATION STRATEGIES 29............................................................................................O
II. AIM OF THE STUDY ..........................................................................32
III. MATERIALS AND METHODS ..........................................................33
USED MATERIALS ....................................................................................................................33
HEMICALS AND MATERIALS 33.........................................................................................................................................C
STANDARDS ................................34MS
NIMALS ...........................................34A
RIMARY AND SECONDARY ANTIBODIES 34.......................................................................................................................P
ABORATORY INSTRUMENTS 35...........L
OFTWARE ........................................36S
LANGENDORFF-PERFUSION OF ISOLATED MOUSE HEARTS ......................................37
BIOANALYTICAL METHODS .................................................................................................37
ROTEIN ASSAY USING BICINCHONINIC ACID (BCA) 37P
WO DIMENSIONAL GEL ELECTROPHORESIS (2-D PAGE) 38.........................................................................................T
SOELECTRICAL FOCUSING (1. DIMENSION) 3..........................................................................................................8I
RADIENT SDS-PAGE (2. D...............................................................................................................9G
ROQ DIAMOND PHOSPHOPROTEIN STAIN 40................................................................................................................P
COMPATIBLE SILVER STAIN........................................................................................................................................MS
OLLOIDAL COOMASSIE STAIN......C
ESTERN BLOT ................................41W
N GEL DIGESTION WITH TRYPSIN 42...I
REPARATION OF HEART SAMPLES FOR COMPARATIVE PHOSPHOPROTEOME ANALYSIS USING STABLE ISOTOPE DIMETHYL P
LABELING ..........................................42
ROTEIN EXTRACTION AND FRACTIONATION FOR IN SOLUTION DIGEST 42..........................................................P
NTERNAL STANDARD – A REFERENCE FOR RELATIVE QUANTITATIVE ANALYSIS..................................................I
EDUCTION AND ALKYLATION OF CYSTEINE RESIDUES 43........................................................................................R
ETHANOL-CHLOROFORM PRECIPITATION OF PROTEINS....................................................................................M
5TABLE OF CONTENTS
ETHANOL PRECIPITATION 43........................................................................................................................................
ROTEIN DIGESTION..................P
TABLE ISOTOPE DIMETHYL LABELING.....................................................................................................................S
AMPLE CLEAN UP USING SOLID PHASE EXTRACTION 44...........................................................................................S
HOSPHOPEPTIDE ENRICHMENT USING TITANIUM DIOXIDE MICRO PARTICLES FILLED TOPTIP 44.........................P
HOSPHOPEPTIDE ENRICHMENT USING CALCIUM PHOSPHATE PRECIPITATION 45..................................................P
AMPLE FRACTIONATION USING SCX FILLED TOPTIP 45.........................................................................................SACE MIXED BED FILLED TOPTIP......................................................................S
NANOLC-ESI-MS ANALYSIS 45...........................................................................................................................................
REPARATION OF A REVERSE PHASE NANOHPLC COLUMN WITH ESI TIP INTERFACE.......................................P
LASS FIBER SOL GEL FRIT 46.........G
NANOETTAN HPLC SETUP FOR REVERSE PHASE CHROMATOGRAPHY 46................................................................
LTIMATE 3000 HPLC SETUP FOR OFF LINE 2D CHROMAT 47U
DATA PROCESSING .....................51MS
ALIBRATION AND TUNING OF THE MASS SPECTOMETER 51.....................................................................................C
SETUP TO ANALYZE PHOSPHORYLATED PEPTIDES 52..........................................................................................LT Q
ORBITRAP ..........................53LT Q
ATABASE SEARCHING 55....................D
ELATIVE QUANTITATION USING QUALBROWSER......................................................................................................R
UANT SETTINGS FOR AUTOMATED QUANTITATION OF PHOSPHOPEPTIDES 56.....................................................MSQ
IV.RESULTS 57..............................................................................................
PRELIMINARY EXPERIMENTS .................................................................