Modeling non-linear dynamic phenomena in biochemical networks [Elektronische Ressource] / vorgelegt von Nicole Radde

Romanohebeler

Sujets

• Mathematics
• Biologie

Informations

• Publié par: universitat_zu_koln
• Publié le: 01 janvier 2007
• Nombre de lectures: 36
• Poids de l'ouvrage: 2 Mo

Extrait

Quantitative proteomics approaches to study leaf senescence
in Arabidopsis thaliana



Dissertation to obtain the degree
Doctor Rerum Naturalium (Dr. rer. nat.)
Of the Faculty of Biology, Ruhr-University Bochum
Department Medical Proteom-Center





submitted by
Romano Hebeler

from Wolfhagen, Germany




Bochum
November 2006


Quantitative proteomische Methoden für die Untersuchung von
Blatt Senescence in Arabidopsis thaliana


Dissertation zur Erlangung des Grades
eines Doktors der Naturwissenschaften
der Fakultät für Biologie
an der Internationalen Graduiertenschule Biowissenschaften
der Ruhr-Universität Bochum


angefertigt am Medizinischen Proteom-Center


vorgelegt von
Romano Hebeler

aus Wolfhagen, Deutschland




Bochum
November 2006 TABLE OF CONTENTS
TABLE OF CONTENTS

1. Introduction ...................................................................................................................... 1
1.1. Arabidopsis thaliana as a model organism to study leaf senescence...................... 1
1.2. Proteomics ............................................................................................................... 4
1.3. Mass spectrometry................................................................................................... 6
1.3.1. Online nano-HPLC/ESI-MS.............................................................................. 7
1.3.2. Quadrupole time-of-flight (QTOF) tandem mass spectrometry........................ 8
1.4. Quantitative proteomic approaches ....................................................................... 10
1.4.1. Protein quantification by densitometric analysis ............................................ 10
1.4.2. Mass spectrometry-based quantitative protein analysis using stable isotope
labeling...........................................................................................................13
1.4.2.1. Chemical derivatization.........................................................................14
1.4.2.2. Metabolic labeling..................................................................................16
1.4.2.3. Interpretation of mass spectra derived from stable isotope coded peptide
pairs....................................................................................................... 19
1.5. Aim of this work...................................................................................................... 20

2. Materials and methods .................................................................................................. 21
2.1. Reagents and chemicals........................................................................................ 21
2.2. Plant material and growth conditions ..................................................................... 21
152.3. N-labeling ............................................................................................................ 21
2.4. Protein extraction and determination of protein concentrations............................. 22
2.5. Labeling of proteins with CyDyes (minimal labeling).............................................. 23
2.6. Separation of proteins by 2-D gel electrophoresis ................................................. 23
2.7. Image acquisition, analysis, and visualization of proteins...................................... 24
2.8. In-gel digestion of proteins..................................................................................... 25
2.9. Chromatographic separation of peptides and mass spectrometry......................... 26
2.10. Analysis of mass spectrometric data ..................................................................... 27
2.11. Determination of protein expression ratios......................................................... 27
2.12. Statistical analyses............................................................................................. 29
2.12.1. Propagation of error model ............................................................................ 29
2.12.2. Box plot analysis ............................................................................................ 29

3. Results........................................................................................................................... 31
3.1. Identification of differentially expressed proteins from wild type and mutant
A. thaliana using DIGE minimal labeling..................................................................31
I TABLE OF CONTENTS
3.2. Relative protein quantification using an advanced strategy composed of DIGE and
15 N-labeling ............................................................................................................ 36
3.2.1. Description of the strategy ............................................................................. 36
153.2.2. Metabolic incorporation of N atoms into proteins of A. thaliana plants........ 39
153.2.3. Linearity of relative protein quantification using N labeling and mass
spectrometry .................................................................................................. 41
14 153.2.4. Evaluation of the elution times of N and N peptides on reversed-phase
nano HPLC..................................................................................................... 42
153.2.5. Evaluation of potential effects of N-labeling on protein expression levels of
A. thaliana ...................................................................................................... 44
153.2.6. Comparison of DIGE versus N-labeling in reversed labeling experiments.. 46

4. Discussion..................................................................................................................... 52
4.1. Proteins differentially expressed in A. thaliana wild type and old1-1 mutant plants...
............................................................................................................................... 52
4.2. Evaluation of protein quantification using an advanced strategy composed of DIGE
15 and N-labeling ..................................................................................................... 55
154.2.1. Metabolic incorporation of N atoms into proteins of A. thaliana plants............
.......................................................................................................................55
154.2.2. Linearity of relative protein quantification using N-labeling and mass
spectrometry..................................................................................................56
14 154.2.3. Co-elution of N/ N-labeled peptide pairs in reversed-phase chromatography57
154.2.4. Evaluation of potential effects of N-labeling on protein expression levels of
A. thaliana ...................................................................................................... 57
154.2.5. Comparison of DIGE vs. N-labeling in reversed labeling experiments........ 58
154.3. Assets and drawbacks of DIGE and N-labeling in quantitative proteomics......... 62
4.4. Future perspectives...............................................................................................65

5. Summary....................................................................................................................... 68

6. Zusammenfassung........................................................................................................69

7. References.................................................................................................................... 70

8. Supplemental information..............................................................................................81

9. Curriculum vitae............................................................................................................. 89
II LIST OF FIGURES AND TABLES
LIST OF FIGURES

Figure 1: Schematic representation of two common proteomic strategies............................ 5

Figure 2: ns of a quadrupole time-of-flight (QTOF) tandem mass
spectrometer. ......................................................................................................... 9

Figure 3: Nomenclature of peptide fragmentation according to Johnson and Martin.......... 10

Figure 4: Labeling reaction of DIGE minimal fluors............................................................. 12

Figure 5: Representative 2-D gel showing protein spots with altered protein concentrations
in A. thaliana wild type and old1-1 mutant plants revealed by DIGE analysis ..... 32

Figure 6: Experimental setup for the identification of differentially expressed proteins in
A. thaliana wild type and old1-1 mutant plants using a double and reverse labeling
approach .............................................................................................................. 37

15Figure 7: Determination of metabolic N incorporation into proteins of A. thaliana wild type
and old1-1 mutant plants via comparison of theoretically and experimentally
acquired peptide mass spetra....................................