PROTEOMICS AND METABOLOMICS INVESTIGATION OF GREEN ALGAE UPON EXPOSURE ...

157 Pages·2016·4 MB·English

by  Gao Yan

PROTEOMICS AND METABOLOMICS
INVESTIGATION OF GREEN ALGAE UPON
EXPOSURE TO PESTICIDES

GAO YAN
(B. Sc Sichuan University)

A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMISTRY
NATIONAL UNIVERSITY OF SINGAPORE
2016
DECLARATION
I hereby declare that this thesis is my original work and it has been written by
me in its entirety, under the supervision of Prof Li Fong Yau, Sam, (in the
laboratory S5-02-05), Chemistry Department, National University of
Singapore, between 01-08-2010 and 10-01-2016.
I have duly acknowledged all the sources of information which have been used
in the thesis.
This thesis has also not been submitted for any degree in any university
previously.
The content of the thesis has been partly published in:
1) Yan Gao, Teck Kwang Lim, Qingsong Lin, Sam Fong Yau Li,
evaluation of sample extraction methods for proteomics analysis of
green algae Chlorella Vulgaris, Electrophoresis, 37(10), 2016, 1270-
1276.
2) Yan Gao, Teck Kwang Lim, Qingsong Lin, Sam Fong Yau Li,
identification of cypermethrin induced proteins changes in green algae
by iTRAQ quantitative proteomics, Journal of Proteomics, 139, 2016,
67-76.

Gao Yan
Name Signature Date
II
Acknowledgement
First and foremost, I would like to express my special appreciation and thanks
to my supervisor, Professor Li Fong Yau, Sam for his valuable guidance and
consistent encouragement I received throughout my candidature. Without his
guidance and support, I could not have completed my thesis.
I would also like to thank Dr Lin Qingsong from the Department of Biological
Sciences for his guidance and support in the proteomics study.
I extend my thanks to my lab mates for providing a warm and engaging
research environment during these years. Many thanks to Mr Lim Teck
Kwang for his technical support and advice in the proteomics study.
Last but not the least, I would like to thank my parents and my brother for
always supporting me spiritually throughout my life.

III
Table of Contents
DECLARATION .............................................................................................. II
Acknowledgement ........................................................................................... III
Table of Contents ............................................................................................. IV
Summary ....................................................................................................... VIII
List of Tables .................................................................................................... X
List of Figures .................................................................................................. XI
List of abbreviations ..................................................................................... XIV
Chapter 1 Introduction ....................................................................................... 1
1.1 Water quality monitoring ......................................................................... 1
1.1.1 General information on water quality monitoring ............................ 1
1.1.2 Biomonitoring of water quality ......................................................... 2
1.1.3 Pesticide pollution ............................................................................. 3
1.2 Proteomics in aquatic toxicology ............................................................. 5
1.2.1 Overview of proteomics .................................................................... 5
1.2.2 Aquatic toxicology proteomics ....................................................... 12
1.3 Metabolomics in aquatic toxicology ...................................................... 14
1.3.1 Overview of metabolomics ............................................................. 14
1.3.2 Aquatic toxicology metabolomics .................................................. 20
1.4 Research scope ....................................................................................... 22
Chapter 2 Evaluation of sample extraction methods for proteomics analysis of
green algae ....................................................................................................... 25
IV
2.1 Introduction ............................................................................................ 25
2.2 Materials and Methods ........................................................................... 27
2.2.1 Microalgae cultivation condition .................................................... 27
2.2.2 Algae protein extraction .................................................................. 28
2.2.3 Protein quantitation and SDS-PAGE analysis ................................ 30
2.2.4 Protein sample preparation ............................................................. 31
2.2.5 Algal proteome profiling using shotgun proteomics ...................... 31
2.2.6 Algal proteome profiling using SWATHTM MS ............................. 33
2.3 Results and Discussion .......................................................................... 34
2.3.1 Quantitative comparison of protein yield ....................................... 34
2.3.2 Shotgun proteomics results ............................................................. 36
2.3.3 SWATH quantitation of proteins .................................................... 39
2.4 Concluding remarks ............................................................................... 41
Chapter 3 Identification of Cypermethrin Induced Proteins Changes in Green
Algae by iTRAQ Quantitative Proteomics ...................................................... 42
3.1 Introduction ............................................................................................ 42
3.2 Materials and Methods ........................................................................... 44
3.2.1 Algae cultivation and cypermethrin treatment ................................ 44
3.2.2 Protein extraction ............................................................................ 45
3.2.3 Protein digestion and iTRAQ labeling ............................................ 46
3.2.4 LC-MS/MS analysis........................................................................ 47
3.2.5 Identification and quantification of peptides and proteins .............. 48
V
3.3 Results and Discussion .......................................................................... 49
3.3.1 Effect of cypermethrin on C. Vulgaris growth ............................... 49
3.3.2 iTRAQ proteomics result overview ................................................ 50
3.3.3 Proteins related with photosynthesis and Calvin cycle ................... 53
3.3.4 Stress responsive proteins ............................................................... 57
3.3.5 Proteins involved with carbohydrates metabolism ......................... 60
3.4. Concluding remarks .............................................................................. 64
Chapter 4 Metabolic response of green algae after exposure to diazinon ....... 69
4.1 Introduction ............................................................................................ 69
4.2 Materials and Methods ........................................................................... 71
4.2.1 Algae cultivation, diazinon treatment and harvest .......................... 71
4.2.2 Sample preparation ......................................................................... 72
4.2.3 LC-MS based metabolic profiling analysis ..................................... 72
4.2.4 Data processing and analysis .......................................................... 73
4.3 Results and Discussion .......................................................................... 74
4.3.1 C. Vulgaris growth .......................................................................... 74
4.3.2 Multivariate analysis of algae samples ........................................... 75
4.3.3 Biochemical mechanism of diazinon toxicity ................................. 79
4.4 Concluding remarks ............................................................................... 85
Chapter 5 Conclusion and future work ............................................................ 86
5.1 Summary of results ................................................................................ 86
VI
5.2 Limitations and future work ................................................................... 88
5.2.1 Measurement of pesticide residues in algae cells ........................... 89
5.2.2 Validation of potential protein and metabolite biomarkers ............ 89
5.2.3 Complementary metabolomics investigation .................................. 90
5.2.4 Exploration of pesticides mixture on algal metabolome and
proteome .................................................................................................. 91
Reference ......................................................................................................... 92
List of Publications ........................................................................................ 106
Appendix ........................................................................................................ 107

VII
Summary
The development of proteomics and metabolomics approaches for evaluation
of pesticides exposure to green algae was explored in this dissertation.
The selection of a proper protein extraction method, which is the very first
step of proteomics study, is of paramount significance for the following
proteomics analysis. Based on this consideration, different protein extraction
methods including direct lysis buffer method, trichloroacetic acid (TCA)
/acetone method, phenol method and phenol-TCA/acetone method were
applied to algae samples to determine the most efficient one. The evaluation of
the above protein extraction methods using a combination of shotgun
proteomics and sequential window acquisition of all theoretical fragment-ion
spectra (SWATH) approach demonstrated that phenol-TCA/acetone method
was the most effective regarding protein yields and protein identifications. The
results offer an insight into the proteomics research on algae species.
Cypermethrin is one of the most widely used pesticides in large scale for
agricultural and domestic purposes and the residue could seriously affect
aquatic system. The determination of the effect of cypermethrin on algal
proteome regarding its exposure concentrations and durations was performed
using isobaric tag for relative and absolute quantitation (iTRAQ) approach. A
total of 162 and 198 proteins were significantly altered after cypermethrin
exposure for 24 h and 96 h in green algae, respectively. The biological
interpretation of these altered proteins indicated that cypermethrin could affect
biological processes in algae including photosynthesis, stress response and
carbohydrate metabolism. This study offers a comprehensive view of the
VIII
complicated modes of action of algal exposure to cypermethrin and provides
several potential protein biomarkers for further investigation of plants and
algae exposed to pollutants in the environment.
The metabolic response of algae to various diazinon concentrations for
different durations was investigated using a liquid chromatography-mass
spectrometry (LC-MS) based metabolomics approach. Multivariate analysis
results showed that diazinon affects algal metabolome in a dosage-dependent
pattern and the discrimination patterns among different groups demonstrated
that algal metabolic changes are more sensitive than growth monitoring,
highlighting the potential of using a metabolomics approach for early toxicity
detection. Diazinon mainly affects metabolites like fatty acids, carbohydrates,
glycerolipids and glycerophospholipids and biological pathways including
lipid metabolism, glycosylphosphatidylinositol-anchor metabolism and
inositol phosphate metabolism in green algae. This study indicates that an LC-
MS based metabolomics approach is a powerful tool for risk assessment of
pesticide toxicity in the environment.

IX
List of Tables
Table 1.1 Advantages and disadvantages of gel based and gel free proteomics
techniques. ................................................................................................. 6
Table 1.2 Common analytical techniques used in metabolomics ................... 16
Table 2.1 Protein yields from green algae using four different extraction
methods. ................................................................................................... 35
Table 2.2. Total number of proteins identified from different protein extraction
methods. ................................................................................................... 37
Table 3.1. Important proteins induced by cypermethrin stress. ....................... 65

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