Hyphenated mass spectrometric methods for quantitative metabolomics in E. coli and human cells [Elektronische Ressource] / vorgelegt von Birgit Timischl

Stephan Strogoff

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 23
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Hyphenated mass spectrometric methods for
quantitative metabolomics in E.coli and human cells


Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)
an der Fakultät für Chemie und Pharmazie
der Universität Regensburg








vorgelegt von
Birgit Timischl
aus Fürstenfeld, Österreich
Juli 2008 Diese Doktorarbeit entstand in der Zeit von April 2004 bis Juni 2008 am Genome
Technology Center der Stanford Universität, Palo Alto, CA, USA, sowie am Institut für
Funktionelle Genomik der Universität Regensburg.

Die Arbeit wurde angeleitet von Prof. Dr. Peter J. Oefner.















Promotionsgesuch eingereicht im Juli 2008

Kolloquiumstermin: 08.09.2008

Prüfungsausschuß: Vorsitzender: Prof. Dr. Manfred Liefländer
Erstgutachter: Prof. Dr. Otto S. Wolfbeis
Zweitgutachter: Prof. Dr. Peter J. Oefner
Drittprüfer: Prof. Dr. Jörg Heilmann 1 Table of Contents
1 TABLE OF CONTENTS............................................................................................................ I
2 ABBREVIATIONS AND ACRONYMS ................................................................................ VI
3 MOTIVATION............................................................................................................................1
4 BACKGROUND .........................................................................................................................6
4.1 METABOLOMICS .......................................................................................................................6
4.2 DETECTION METHODS FOR METABOLOMICS..........................................................................9
4.2.1 MASS SPECTROMETRY...........................................................................................................10
4.2.2 NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY (NMR) ..................................................15
4.3 METHODS FOR METABOLOME ANALYSIS..............................................................................17
4.3.1 DIRECT INFUSION ..................................................................................................................17
4.3.2 MATRIX-ASSISTED LASER DESOPTION/IONIZATION – MASS SPECTROMETRY (MALDI-MS)18
4.3.3 CAPILLARY ELECTROPHORESIS.............................................................................................18
4.3.4 LIQUID CHROMATOGRAPHY ..................................................................................................20
4.3.5 GAS CHROMATOGRAPHY.......................................................................................................28
4.3.6 COMPARISON OF METHODS FOR THE ANALYSIS OF CENTRAL CARBON METABOLITES .........31
4.3.7 FLUX ANALYSIS.....................................................................................................................34
4.3.8 PLATFORM APPROACHES.......................................................................................................35
4.4 SAMPLE PREPARATION...........................................................................................................36
4.5 DATA ANALYSIS.......................................................................................................................38
4.5.1 HANDLING OF COMPLEX METABOLOMIC DATASETS.............................................................38
4.5.2 QUANTIFICATION ..................................................................................................................40
5 EXPERIMENTAL SECTION – MATERIALS AND INSTRUMENTATION ..................41
5.1 CHEMICALS.............................................................................................................................41
5.2 INSTRUMENTATION ................................................................................................................41
5.3 SOFTWARE...............................................................................................................................42
I6 CE-TOF MS METHOD DEVELOPMENT ...........................................................................43
6.1 INTRODUCTION .......................................................................................................................43
6.2 METHODS ................................................................................................................................43
6.2.1 EXPERIMENTAL DETAILS OF THE OPTIMIZED CE-TOF-MS METHOD....................................43
6.3 RESULTS AND DISCUSSION .....................................................................................................45
6.3.1 CHOICE OF CAPILLARY COATING ..........................................................................................45
6.3.2 OPTIMIZATION OF THE BACKGROUND ELECTROLYTE...........................................................45
6.3.3 SHEATH LIQUID OPTIMIZATION .............................................................................................46
6.3.4 MIGRATION TIME SHIFT.........................................................................................................48
6.3.5 QUANTITATIVE METABOLITE ANALYSIS AND QUALITY CONTROL........................................49
7 OPTIMIZATION OF SAMPLING AND METHOD VALIDATION FOR ESCHERICHIA
COLI .................................................................................................................................................52
7.1 INTRODUCTION .......................................................................................................................52
7.2 MATERIAL AND METHODS.....................................................................................................52
7.2.1 BACTERIAL STRAINS AND GROWTH CONDITIONS..................................................................52
7.2.2 EXPERIMENTAL DETAILS FOR CELL HARVESTING AND METABOLITE EXTRACTION..............53
7.2.3 CE-TOF-MS..........................................................................................................................54
7.3 RESULTS AND DISCUSSION .....................................................................................................54
7.3.1 OPTIMIZATION OF CELL HARVESTING AND METABOLITE EXTRACTION................................54
7.3.2 QUANTIFICATION IN BIOLOGICAL MATRICES ........................................................................56
8 METABOLOME ANALYSIS OF E. COLI............................................................................58
8.1 INTRODUCTION .......................................................................................................................58
8.2 MATERIAL AND METHODS.....................................................................................................59
8.2.1 BACTERIAL STRAINS, GROWTH CONDITIONS AND SAMPLE EXTRACTION .............................59
8.2.2 CE-TOF-MS..........................................................................................................................59
8.2.3 GAS CHROMATOGRAPHY-MASS SPECTROMETRY..................................................................59
8.2.4 GC-MS OF AMINO ACIDS ......................................................................................................60
8.2.5 DATA ANALYSIS FOR FEATURE DETECTION ..........................................................................60
8.3 RESULTS AND DISCUSSION .....................................................................................................61
II8.3.1 METABOLIC PROFILING IN E. COLI - METHOD VALIDATION BY COMPARISON TO OTHER
ANALYTICAL METHODS .....................................................................................................................61
8.3.2 EVALUATION OF THE ROBUSTNESS OF OBSERVED DIFFERENCES IN METABOLITE LEVELS...65
8.3.3 METABOLIC FINGERPRINTING OF E. COLI .............................................................................67
8.3.4 THE VALIDATED CE-TOF-MS METHOD IN THE CONTEXT OF RECENT DEVELOPMENTS IN
METABOLOME ANALYSIS...................................................................................................................72
9 METHOD DEVELOPMENT FOR RAPID LACTATE MEASUREMENT......................73
9.1 INTRODUCTION .......................................................................................................................73
9.2 MATERIAL AND METHODS.....................................................................................................74
9.2.1 IP-LC-MS/MS.......................................................................................................................74
9.3 RESULTS AND DISCUSSION .....................................................................................................75
9.3.1 METHOD DEVELOPMENT FOR FAST LACTATE MEASUREMENT..............................................75
9.3.2 UPTAKE OF LACTIC ACID INTO DIFFERENT IMMUNE CELL POPULATIONS .............................76
10 METHOD DEVELOPMENT FOR TARGETED METABOLITE ANALYSIS USING
RAPID RESOLUTION IP-LC-MS/MS ........................................................................................78
10.1 INTRODUCTION ..................................................