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Applicability of proteomics to assess effects due to genetic engineering in the context of natural variability using Arabidopsis thaliana as a model organism [Elektronische Ressource] / Martin Christian Rübelt

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Published 01 January 2005
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TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Allgemeine Lebensmitteltechnologie






Applicability of proteomics to assess effects due to genetic engineering in
the context of natural variability using Arabidopsis thaliana as a
model organism


Martin Christian Rübelt


Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für
Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung
des akademischen Grades eines


Doktors der Naturwissenschaften
(Dr. rer. nat.)


genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. Gerhard Wenzel
Prüfer der Dissertation: 1. Univ.-Prof. Dr. Karl-Heinz Engel
2. apl. Prof. Dr. Angelika Görg
3. apl. Prof. Dr. Klaus-Dieter Jany, Universität Stuttgart


Die Dissertation wurde am 17.12.2004 bei der Technischen Universität München eingereicht
und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung
und Umwelt am 21.01.2005 angenommen.


Acknowledgements
I am most grateful to the members of my dissertation committee: Professor Dr. Klaus-Dieter
Jany for inspiring my interest in the topic, helping to make possible a scholarship at
Monsanto Company, ensuring that I stay on the right track with personal interest and
encouragement at every step of the work; Professor Dr. Karl-Heinz Engel for valuable
guidance, strategic direction, and critical review of my thesis; Professor Dr. Angela Görg for
kindly agreeing to serve as a committee member and for evaluating my work in light of her
extensive expertise in proteomics; and Professor Dr. Gerhard Wenzel for chairmaning the
dissertation committee.
I am also deeply indebted to my advisor team at the Monsanto Company, St. Louis: my
supervisor James Astwood, PhD, for consistent confidence in my work, his enthusiasm in my
project and regular interest in its progress, as well as useful advice on thesis related issues;
Markus Lipp, PhD, for his professional input on method validation and on other crucial
aspects of the work, and stimulating my creativity and motivation with his friendship and
support; Tracey Reynolds, PhD, for dedicating her time and effort to review countless
versions of my thesis, providing insightful comments on content, style, and presentation (in
other words, for teaching me how to write), and for the overall unfaltering interest in the
progress of my work; and Cherian George, PhD, for assistance in the early stages of this
work.
The completion of this thesis would also not be possible without the following individuals:
Margaret Nemeth, PhD, who provided advice on statistics and experimental design, and
always patiently explaining complex concepts to an incredibly thickheaded individual like
me; Nancy Leimgruber, who introduced me to the dark art of 2DE and always welcomed my
presence in her laboratory; Richard Thoma, who deepened my skills in mass spectroscopy;
Bala Karunanandaa, PhD, who opened my eyes to the model plant Arabidopsis thaliana,
which I mistakenly considered "an uninteresting weed"; Kanthasamy (Karu) Karunanandaa,
PhD, who kindly took me under his wing and brought me in touch with various teams and
people at Monsanto, which proved crucial for the successful completion of my work; Daniel
Free, who assisted me with the cultivation of the Arabidopsis plants; Professor Dean
DellaPenna, PhD, of Michigan State University and Jon Schmuke, who provided me with the
transgenic Arabidopsis lines and characterization data; and all the employees of Regulatory
Science, Crop Analytics, Biotechnology, and Library Services at Monsanto Company, St.
Louis, who contributed in numerous ways to this work and made me feel comfortable during
its various stages.
Finally, I am grateful to Monsanto Company for providing generous financial assistance and
access to top-notch facilities for my research in the form of a three-year scholarship. This
amazing opportunity has given me incredible inspiration for my future endeavors as a
scientist and development as a person.











This thesis is dedicated to my parents
Dr. Christian Rübelt and Doris Rübelt.

Table of contents Page 1

1 Introduction and aim of study........................................................................................ 5
2 Background ......................................................................................................................7
2.1 Biotechnology............................................................................................................7
2.2 Regulation of genetically modified organisms (GMOs) .......................................... 10
2.2.1 EU regulation of genetically modified organism/food........................................ 12
2.3 Safety assessment of genetically modified food ...................................................... 15
2.4 Unintended effect(s).................................................................................................18
2.5 Profiling techniques..................................................................................................24
2.5.1 Transcriptomics...................................................................................................
2.5.2 Proteomics...........................................................................................................26
2.5.3 Metabolomics......................................................................................................29
2.6 2DE-based proteomics.............................................................................................31
2.6.1 2DE: State of the art............................................................................................ 32
2.6.1.1 Sample preparation......................................................................................34
2.6.1.2 First dimension: IEF with immobilized pH gradients................................. 36
2.6.1.3 Second dimension: SDS-PAGE .................................................................. 38
2.6.1.4 Detection of proteins................................................................................... 39
2.6.1.5 Image acquisition and analysis ................................................................... 42
2.6.1.6 Protein identification43
3 Materials and methods .................................................................................................. 45
3.1 Materials................................................................................................................... 45
3.1.1 Plant lines............................................................................................................45
3.1.1.1 A. thaliana ecotypes.................................................................................... 45
3.1.1.2 Transgenic A. thaliana lines........................................................................ 45
3.1.2 Chemicals45
3.1.3 Equipment...........................................................................................................46
3.1.4 Software..............................................................................................................47
3.2 Methods.................................................................................................................... 47
3.2.1 Growth of plants.................................................................................................. 47
3.2.2 Harvesting48
3.2.3 Storage................................................................................................................48
3.2.4 Phenotypic analysis and methodology................................................................48
3.2.5 Protein extraction................................................................................................48
3.2.5.1 Extraction buffers........................................................................................49
3.2.5.2 Mortar and pestle grinding method.............................................................
3.2.5.3 Mega Grinder method ................................................................................. 50
3.2.5.4 Defatting of seed ......................................................................................... 50
3.2.5.5 Protein quantification..................................................................................50
3.2.6 IEF-PAGE...........................................................................................................51
3.2.7 Two-dimensional gel electrophoresis (2DE) ...................................................... 51
Page 2 Table of contents

3.2.7.1 First dimension............................................................................................51
3.2.7.2 Second dimension........................................................................................
3.2.7.3 Molecular weight standards.........................................................................52
3.2.7.4 Staining methods.........................................................................................52
3.2.7.4.1 Silver stain...............................................................................................
3.2.7.4.2 Fluorescent stain.....................................................................................52
3.2.7.4.3 Colloidal Coomassie Brilliant Blue ........................................................53
3.2.7.5 Image analysis.............................................................................................54
3.2.7.5.1 Spot detection..........................................................................................
3.2.7.5.2 Protein expression comparison...............................................................54
3.2.7.5.3 Mw and pI calibration.............................................................................55
3.2.8 Protein identification by MALDI-TOF MS.........................................................56
3.2.8.1 In-gel tryptic digestion.................................................................................56
3.2.8.2 MALDI-TOF analysis57
3.2.9 Phenetic tree.........................................................................................................
4 Result and Discussion.....................................................................................................58
4.1 2DE method development and optimization.............................................................58
4.1.1 Grinding...............................................................................................................58
4.1.2 Protein extraction.................................................................................................61
4.1.2.1 Defatting of seeds prior protein extraction ..................................................62
4.1.2.2 Extraction temperature.................................................................................63
4.1.2.3 Extraction buffer..........................................................................................
4.1.3 Isoelectric focusing (IEF) ....................................................................................69
4.1.3.1 Choice of the pH gradient............................................................................69
4.1.3.2 Sample application.......................................................................................71
4.1.3.3 Isoelectrofocusing program.........................................................................
4.1.4 Second dimension (SDS-PAGE)72
4.1.4.1 Choice of the SDS-PAGE gel gradient........................................................72
4.1.5 Staining................................................................................................................74
4.1.5.1 Silver staining..............................................................................................74
4.1.5.2 Coomassie Brilliant Blue staining (CBB)....................................................76
4.1.5.3 Fluorescent staining.....................................................................................77
4.1.6 Summary..............................................................................................................79
4.2 Validation of the 2DE method ..................................................................................81
4.2.1 Repeatability........................................................................................................81
4.2.2 Estimation of replicate size89
4.2.3 Sensitivity and linearity of response....................................................................92
4.2.3.1 Sensitivity....................................................................................................94
4.2.3.2 Linearity.......................................................................................................96
4.2.4 Summary99
4.3 Natural variation.....................................................................................................101
4.3.1 Environmental....................................................................................................102
4.3.1.1 Plant-to-plant variability............................................................................103
4.3.1.1.1 Comparison of phenotypic traits...........................................................
4.3.1.1.2 Comparison of 2DE pattern ..................................................................105
4.3.1.2 Effect of different environments................................................................107
4.3.2 Genetic background...........................................................................................113
Table of contents Page 3

4.3.2.1 Comparison of phenotypic traits ............................................................... 114
4.3.2.2 Comparison of 2DE patterns..................................................................... 115
4.3.2.2.1 Qualitative comparison of 2DE patterns .............................................. 116
4.3.2.2.2 Quantitative comp ............................................ 120
4.3.2.2.3 Quantification of the natural variation ................................................. 122
4.3.3 Summary...........................................................................................................125
4.4 Genetically modified lines......................................................................................127
4.4.1 GUS-lines..........................................................................................................
4.4.1.1 Phenotypic comparison.............................................................................129
4.4.1.2 Comparison of 2DE patterns..................................................................... 130
4.4.1.2.1 Qualitative comparison of 2DE patterns .............................................. 130
4.4.1.2.2 Identification of protein spot SSP 2814................................................ 131
4.4.1.3 Quantitative comparison...........................................................................133
4.4.1.3.1 Quantitative comparison of the novel protein ......................................
4.4.1.3.2 parison of 2DE patterns ............................................ 134
4.4.2 Enhanced tocopherol lines................................................................................139
4.4.2.1 Phenotypic comparison140
4.4.2.2 Comparison of 2DE patterns..................................................................... 142
4.4.2.2.1 Qualitative comparison of 2DE pattern................................................ 143
4.4.2.2.2 Quantitative comp.............................................. 144
4.4.3 Summary...........................................................................................................147
5 Summary ...................................................................................................................... 148
6 Zusammenfassung ....................................................................................................... 150
7 Bibliography................................................................................................................. 152
Appendix 1: Spreadsheet for the Jaccard index calculation........................................... 174
Appendix 2: Ecotype study - Quantitative data (IOD).................................................... 176
Curriculum Vitae ................................................................................................................ 195

Page 4 Abbreviations

Abbreviations

2DE two-dimensional gel electrophoresis
ADI acceptable daily intake
BSA Bovine Serum Albumin
CBB Coomassie Brilliant Blue
cDNA complementaryDNA
CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
CV coefficient of variation
CYMAL cyclohexyl-b-D-maltoside
DNA deoxyribonucleic acid
DNase I deoxyrlease I
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
EGTA ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid
FAO Food and Agriculture Organization of the United Nations
FDA d Drug Administration
FOS-Choline-10 n-dodecylphosphosphocholine
fw fresh weight
g gravitational acceleration
ID identification
IEF isoelectric focusing
IOD integrated optical density
IPG immobilized pH gradient
kD kilo Dalton
MALDI matrix-assisted-desorption-ionization
MS mass spectrometry
Mw molecular weight
N sample size
OD optical density
PAGE polyacrylamide gel electrophoresis
PBS Phosphate Buffered Saline
pI isoelectric point
PMF peptide mass fingerprinting
PMSF phenylmethanesulfonylfluoride
R2 coefficient of determination for line regression
RNA ribonucleic acid
RNase A ribonuclease A.
rpm revolution per minute
RT room temperature
SD standard deviation
SDS sodium dodecyl sulfate
TBP tributylphosphine
TCA trichloroacetic acid
TCEP Tris(2-carboxyethyl)phosphine
T-DNA transfer DNA
TOF time-of-flight
Vh volt-hours
WHO World Health Organization