UNIVERSITI SAINS MALAYSIA 2010eprints.usm.my/42578/1/SHIM_SIANG_YIAN.pdf · UNIVERSITI SAINS...
Transcript of UNIVERSITI SAINS MALAYSIA 2010eprints.usm.my/42578/1/SHIM_SIANG_YIAN.pdf · UNIVERSITI SAINS...
ANALYSIS OF PROTEINS EXTRACTED FROM THE LEAVES
OF Piper sarmentosum
SHIM SIANG YIAN
UNIVERSITI SAINS MALAYSIA
2010
ANALYSIS OF PROTEINS EXTRACTED FROM THE LEAVES
OF Piper sarmentosum
by
SHIM SIANG YIAN
Thesis submitted in fulfillment of the
requirements for the degree of
Master of Sceince
October 2010
ii
ACKNOWLEDGEMENT
Firstly, I would like to extend my gratitude to my supervisor, Assoc. Prof. Dr
Gam Lay Harn for her help and guidance throughout my study. I would also like to
thank my co-supervisors Prof. Dr. Zhari for his assistance and experience during my
study.
I would like to extend my gratitude to the staffs of the School of Pharmaceutical
Sciences and the National Poison Centre for assisting me by providing facilities,
equipment and assistance to my work.
I would like to thank Mr. Shumugan for Pusat Pengajian Kajihayat for plant
identification.
Finally, I would like to thank my family members and my labmates for their
assistance, advice and support.
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TABLE OF CONTENTS
Page
Acknowledgement ii
Table of Contents iii
List of Tables ix
List of Figures x
List of Abbreviations xix
List of Appendices xxi
Abstrak xxii
Abstract xxiv
CHAPTER 1 – INTRODUCTION 1
1.1.1 Traditional Medicinal Plants 1
1.2 Piper 2
1.3 Piper sarmentosum 3
1.4 Protein, Polypeptide, Peptide and Amino Acid 4
1.5 Proteomics 6
1.6 Plant Proteomics 7
1.7 Sample Preparation 11
1.8 Protein Extraction Buffer 14
1.9 Protein Precipitation and Purification 16
1.10 Protein Assay 16
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1.11 Two-Dimensional Gel Electrophoresis 17
1.11.1 Isoelectric Focusing 18
1.11.2 Sodium Dodecyl Sulfate Polyacrylamide Gel 19
Electrophoresis (SDS-PAGE)
1.12 Staining 20
1.12.1 Coomassie Brilliant Blue-R 21
1.12.2 Coomassie Brilliant Blue-G 22
1.13 In-gel Digestion 22
1.13.1 Trypsin 23
1.14 Liquid Chromatography- Mass Spectrometry 24
1.14.1 Reverse-phased High Performance Liquid 24
Chromatography (RP-HPLC)
1.14.1.1 Pump 26
1.14.1.2 Injector 26
1.14.1.3 Mobile Phase 26
1.14.1.4 Column 28
1.14.2 Mass Spectrometry 28
v
1.14.2.1 Ionization Source 29
1.14.2.2 Mass Analyzer 29
1.14.2.3 Tandem Mass Spectrometry 30
1.15 Mascot Search Engine and Bioinformatics in Proteomic 31
Analysis
1.16 Western Blotting 33
1.17 Research Objectives 34
CHAPTER 2 – METHODS AND MATERIALS 35
2.1 Chemicals and Reagents 35
2.2 Method Development 35
2.2.1 Plant Cells Disruption Methods 35
2.2.2 Comparison of Extraction Methods 37
2.2.3 TCA/Acetone precipitation 38
2.2.4 Protein Assay 39
2.2.5 SDS-PAGE 40
2.2.6 2-DE 40
2.3 Protein Analysis of Leaf Extracts of P. sarmentosum 41
vi
2.3.1 Herbal Samples 41
2.3.2 Protein Extraction 42
2.3.3 TCA/Acetone precipitation 42
2.3.4 Protein Assay 42
2.3.5 2-DE 42
2.3.6 Image Analysis of 2-DE gel 44
2.3.7 In-Gel Digestion 44
2.3.8 Mass Spectrometric Analysis 45
2.3.9 Protein Identification 46
2.3.10 Western Blotting 46
CHAPTER 3 – RESULTS 48
3.1 Method Development 48
3.1.1 Plant Cells Disruption Methods 48
3.1.2 Comparison of Extraction Methods 52
3.1.2.1 Comparison of Extraction Methods Using SDS-PAGE 52
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3.1.2.2 Comparison of Extraction Methods Using 54
2-DE Technique
3.1.3 Evaluation of pH range of IPG strip 60
3.2 Protein Profiling of Leaves of P. sarmentosum From FRIM 62
3.2.1 Sequence Alignment of Protein 94
3.2.2 Cellular Localization of Proteins 100
3.2.3 Functional Classification of Proteins 102
3.3 Protein Analysis of P. sarmentosum 104
3.3.1 Evaluation of 2-DE Protein Spot 104
3.3.2 Comparison of Proteome of Leaf Extracts from 122
Different Geographical Areas
3.4 Common Protein Spots with Differential Protein Expression 153
According to Geographical Areas
3.4.1 Comparison of Protein Spot from Young Leaf Extracts of 153
P. sarmentosum from Different geographical Areas
3.4.2 Comparison of Mature Leaf Protein Spot of P. sarmentosum 161
from Different Geographical Areas
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3.5 Unique Protein Spots 169
3.6 Irregular Protein Spot 173
3.7 Western Blotting 188
CHAPTER 4 – DISCUSSION 189
4.1 Method Development 189
4.1.1 Plant Cell Disruption Methods 189
4.1.2 Comparison of Extraction Methods 192
4.2 Protein Profiling of P. sarmentosum 197
4.3 Protein Analysis of Leaf Extracts of P. sarmentosum 203
4.4 Application of Identified Proteins in Future Study 207
CHAPTER 5 – CONCLUSION 208
REFERENCES 209
APPENDICES 222
LIST OF PUBLICATIONS 310
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LIST OF TABLES
Page
Table 3.1 List of identified proteins in the leaf extracts of 67
P. sarmentosum.
Table 3.2 High intense protein spots identification (ppm> 10,000) in 127
young and mature leaf extracts of P. sarmentosum.
Table 3.3 Medium intense protein spots identification (ppm 2000-8000) 142
in young and mature leaf extracts of P. sarmentosum.
Table 3.4 Lower intense protein spots identification (ppm< 2,000) in 150
young and mature leaf extracts of P. sarmentosum.
Table 3.5 Differential protein expression on common spot proteins 156
from the young leaf extracts of P. sarmentosum in seven
geographical areas.
Table 3.6 Differential protein expression on common spot proteins 164
from the mature leaf extracts of P. sarmentosum in seven
geographical areas.
Table 3.7 Unique protein spot images from the young and mature leaf 170
extracts of P. sarmentosum.
Table 3.8 Identification of proteins from the unique protein spots of 172
young and mature leaf extracts of P. sarmentosum
Table 3.9 Irregular protein spot images from the young and mature 174
leaf extracts of P. sarmentosum
Table 3.10 Absent protein spot images for young and mature leaf extracts 185
of P. sarmentosum
x
LIST OF FIGURES
Page
Figure 3.1 Protein profiles by SDS-PAGE of the leave of 49
P. sarmentosum using different extraction methods.
Each sample loaded and ran in three wells for a) and
b) and two wells for c) and d). a) Blending combined
with ultrasonic disruption method. b) Blending combined
with bead beating method. c) Combination of glass bead
and grinding method. d) Grinding method with a mortal
and pestle in the presence of liquid nitrogen.
Figure 3.2 Protein profiles of the leave of P. sarmentosum using 50
different cell disruption methods. a) 5 protein bands were
detected using blending combined with ultrasonic
disruption method. b) 7 protein bands were detected using
blending combined with bead beating method. c) 15 protein
bands were detected using a combination of glass bead and
grinding method. d) 20 protein bands were detected using
grinding method with a mortar and pestle in the presence
of liquid nitrogen.
Figure 3.3 Protein profiles of the leave of P. sarmentosum using 51
cell disruption method of grinding with liquid nitrogen
method. Each sample loaded and ran in three wells. a) 8.7g
leaf weight on SDS-PAGE gels. b) 26 protein bands were
detected using 8.7g of leaf weight on SDS-PAGE gels.
Figure 3.4 Protein profiles of the leave of P. sarmentosum using 53
different extraction methods. M: marker; Lane 1: Tris
extraction buffer in the first stage of the sequential
extraction method followed by TCA/acetone precipitation.
Lane 2: TLB extraction buffer in the second stage of the
sequential extraction method followed by TCA/acetone
precipitation. Lane 3: protein bands detection in Lane 1.
Lane 4: protein bands detection in Lane 2. Lane 5: TLB
extraction buffer in total protein extraction method followed
by TCA/acetone precipitation. Lane 6: TCA/acetone wash
prior to TLB extraction method and followed by
TCA/acetone precipitation. Lane 7: protein bands detection
in Lane 5. Lane 8: protein bands detection in Lane 6.
Figure 3.5 Protein profiles by 2-DE gel of the leave of P. sarmentosum 56
using different extraction methods. a) Tris extraction buffer
in the first stage of the sequential extraction method
followed by TCA/acetone precipitation. b) TLB extraction
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buffer in the second stage of the sequential extraction
method followed by TCA/acetone precipitation. c) TLB
extraction buffer in total protein extraction method followed
by TCA/acetone precipitation. d) TLB extraction buffer in
TCA/acetone wash prior to TLB extraction method and
followed by TCA/acetone precipitation.
Figure 3.6 Protein spot detection on 2-DE gel from the leave of 57
P. sarmentosum using different extraction methods.
Each circle represents a protein spot. a) 153 protein spots
were detected using Tris extraction buffer in the first stage
of sequential extraction method. b) 135 protein spots were
detected using TLB extraction buffer in the second stage of
sequential extraction method. c) 187 protein spots were
detected using TLB extraction buffer in total protein extraction
method. d) 133 protein spots were detected using TLB
extraction buffer in TCA/acetone wash prior to TLB extraction
method and followed by TCA/acetone precipitation.
Figure 3.7 Comparison of protein profiles of 2-DE gel of leaves of 58
P. sarmentosum using total protein extraction method and
sequential extraction methods. a) 58 unique protein spots
using TLB extraction buffer in total protein extraction
method. b) 13 unique protein spots using Tris extraction
buffer in the first stage of the sequential extraction method.
c) 7 unique protein spots using TLB extraction buffer in the
second stage of the sequential extraction method. d) The
number of the common spots and unique spots between
the total protein extraction method and sequential
extraction method.
Figure 3.8 Comparison of protein profiles of 2-DE gel of the leaves of 59
P. sarmentosum using total protein extraction method and
TCA/acetone wash prior to TLB extraction method and
followed by TCA/acetone precipitation. a) Unique proteins
using TLB extraction buffer in total protein extraction
method. b) Unique proteins using TLB extraction buffer
in TCA/acetone wash prior to TLB extraction method and
followed by TCA/acetone precipitation. c) The number of
the common spots and unique spots between the total protein
extraction method and TCA/acetone wash prior to TLB
extraction method and followed by TCA/acetone precipitation.
Figure 3.9 a) Leaf protein profiles of P. sarmentosum analyzed by 61
2-DE on 3-10 pH range IPG strip. b) Leaf protein profiles
of P. sarmentosum analyzed by 2-DE on 4-7 pH range IPG strip.
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Figure 3.10 2-DE gel image with the excised protein spots (circled) for 64
LCMS/MS analysis.
Figure 3.11 Distribution of proteins in 2-DE gel of the leaf extracts of 101
P. sarmentosum according subcellular localization.
Figure 3.12 Distribution of proteins in 2-DE gel of the leaf extracts of 103
P. sarmentosum according to functional classification.
Figure 3.13 a) Protein profiles by 2-DE gel of the young leaves of 106
P. sarmentosum from USM, Penang. b) 185 protein spots
were detected in the 2-DE gel of the young leaves of
P. sarmentosum from USM, Penang. c) Protein profiles
of 2-DE gel of the young leaves of P. sarmentosum from
USM, Penang. d) 185 protein spots were detected in the
2-DE gel of the young leaves of P. sarmentosum from
USM, Penang.
Figure 3.14 a) Protein profiles by 2-DE gel of the young leaves of 107
P. sarmentosum from Kepala Batas, Penang. b) 219 protein
spots were detected in the 2-DE gel of the young leaves of
P. sarmentosum from Kepala Batas, Penang. c) Protein
profiles of 2-DE gel of the young leaves of
P. sarmentosum from Kepala Batas, Penang. d) 218
protein spots were detected in the 2-DE gel of the young
leaves of P. sarmentosum from Kepala Batas, Penang.
Figure 3.15 a) Protein profiles by 2-DE gel of the young leaves of 108
P. sarmentosum from Botanical Garden, Penang. b) 181
protein spots were detected in the 2-DE gel of the young
leaves of P. sarmentosum from Botanical Garden, Penang.
c) Protein profiles of 2-DE gel of the young leave of
P. sarmentosum from Botanical Garden, Penang. d) 178
protein spots were detected in the 2-DE gel of the young
leaves of P. sarmentosum from Botanical Garden, Penang.
Figure 3.16 a) Protein profiles by 2-DE gel of the young leaves of 109
P. sarmentosum from Sarawak. b) 226 protein spots
were detected in the 2-DE gel of the young leaves of
P. sarmentosum from Sarawak. c) Protein profiles of 2-DE
gel of the young leaves of P. sarmentosum from Sarawak.
d) 224 protein spots were detected in the 2-DE gel of the
young leaves of P. sarmentosum from Sarawak.
Figure 3.17 a) Protein profiles by 2-DE gel of the young leaves of 110
P. sarmentosum from Johor. b) 206 protein spots were
detected in the 2-DE gel of the young leaves of
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P. sarmentosum from Johor. c) Protein profiles of 2-DE
gel of the young leaves of P. sarmentosum from Johor.
d) 206 protein spots were detected in the 2-DE gel of the
young leaves of P. sarmentosum from Johor.
Figure 3.18 a) Protein profiles by 2-DE gel of the young leaves of 111
P. sarmentosum from Kuala Lumpur. b) 191 protein
spots were detected in the 2-DE gel of the young leaves of
P. sarmentosum from Kuala Lumpur. c) Protein profiles
of 2-DE gel of the young leaves of P. sarmentosum from
Kuala Lumpur d) 191 protein spots were detected in the
2-DE gel of the young leaves of P. sarmentosum from
Kuala Lumpur.
Figure 3.19 a) Protein profiles by 2-DE gel of the young leaves of 112
P. sarmentosum from Kedah. b) 207 protein spots were
detected in the 2-DE gel of the young leaves of
P. sarmentosum from Kedah. c) Protein profiles of 2-DE
gel of the young leaves of P. sarmentosum from Kedah.
d) 208 protein spots were detected in the 2-DE gel of the
young leaves of P. sarmentosum from Kedah.
Figure 3.20 a) Protein profiles by 2-DE gel of the mature leaves of 114
P. sarmentosum from USM, Penang. b) 141 protein spots
were detected in the 2-DE gel of the mature leaves of
P. sarmentosum from USM, Penang. c) Protein profiles
of 2-DE gel of the mature leaves of P. sarmentosum from
USM, Penang. d) 138 protein spots were detected in the
2-DE gel of the mature leaves of P. sarmentosum from
USM, Penang.
Figure 3.21 a) Protein profiles by 2-DE gel of the mature leaves of 115
P. sarmentosum from Kepala Batas, Penang. b) 184 protein
spots were detected in the 2-DE gel of the mature leaves of
P. sarmentosum from Kepala Batas, Penang. c) Protein
profiles of 2-DE gel of the mature leaves of
P. sarmentosum from Kepala Batas, Penang. d) 184 protein
spots were detected in the 2-DE gel of the mature leaves of
P. sarmentosum from Kepala Batas, Penang.
Figure 3.22 a) Protein profiles by 2-DE gel of the mature leaves of 116
P. sarmentosum from Botanical Garden, Penang. b) 181
protein spots were detected in the 2-DE gel of the mature
leaves of P. sarmentosum from Botanical Garden, Penang.
c) Protein profiles of 2-DE gel of the mature leaves of
P. sarmentosum from Botanical Garden, Penang. d) 181
protein spots were detected in the 2-DE gel of the mature
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leaves of P. sarmentosum from Botanical Garden, Penang.
Figure 3.23 a) Protein profiles by 2-DE gel of the mature leaves of 117
P. sarmentosum from Sarawak. b) 188 protein spots were
detected in the 2-DE gel of the mature leaves of
P. sarmentosum from Sarawak. c) Protein profiles of
2-DE gel of the mature leaves of P. sarmentosum from
Sarawak. d) 187 protein spots were detected in the
2-DE gel of the mature leaves of P. sarmentosum from
Sarawak.
Figure 3.24 a) Protein profiles by 2-DE gel of the mature leaves of 118
P. sarmentosum from Johor. b) 148 protein spots were
detected in the 2-DE gel of the mature leaves of
P. sarmentosum from Johor. c) Protein profiles of 2-DE
gel of the mature leaves of P. sarmentosum from Johor.
d) 148 protein spots were detected in the 2-DE gel of the
mature leaves of P. sarmentosum from Johor.
Figure 3.25 a) Protein profiles by 2-DE gel of the mature leaves of 119
P. sarmentosum from Kuala Lumpur. b) 189 protein
spots were detected in the 2-DE gel of the mature leaves
of P. sarmentosum from Kuala Lumpur. c) Protein profiles
of 2-DE gel of the mature leaves of P. sarmentosum
from Kuala Lumpur. d) 186 protein spots were detected in
the 2-DE gel of the mature leaves of P. sarmentosum from
Kuala Lumpur.
Figure 3.26 a) Protein profiles by 2-DE gel of the mature leaves of 120
P. sarmentosum from Kedah. b) 169 protein spots were
detected in the 2-DE gel of the mature leaves of
P. sarmentosum from Kedah. c) Protein profiles of
2-DE gel of the mature leaves of P. sarmentosum from
Kedah. d) 166 protein spots were detected in the 2-DE gel
of the mature leaves of P. sarmentosum from Kedah.
Figure 3.27 Five landmarks had been selected in the protein 121
analysis of leaf extracts of P. sarmentosum.
Figure 3.28 84 common protein spots (circled) in the 2-DE gel of each 125
young and mature leaves of P. sarmentosum from seven
geographical areas.
Figure 3.29 The 16 significant high intense common protein spots 126
identified from the 2-DE gel of young and mature leaf
extracts of P. sarmentosum from seven geographical areas.
Spot # 1 was homology protein with a Mowse score below 44.
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Figure 3.30 Mascot search engine for ATP synthase beta subunit which 129
had Mowse score 639 and sequence coverage 25%.
Nine peptide sequences identified by MS/MS were
underlined.
Figure 3.31 Mass spectrometry analysis for protein identification. 130
a) Base peak chromatogram; b) MS scan at 45.4 min
showing one of the most intense precursor ion, 978.1 m/z
which was subjected to MS/MS scan; c) MS/MS spectrum;
d) amino acid sequence derived from MS/MS spectra
which belong to ATP synthase beta subunit.
Figure 3.32 a) MS scan at 40.0 min showing one of the most intense 131
precursor ion, 596.3 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.33 a) MS scan at 42.6 min showing one of the most intense 132
precursor ion, 504.4 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.34 a) MS scan at 41.0 min showing one of the most intense 133
precursor ion, 488.3 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.35 a) MS scan at 51.7 min showing one of the most intense 134
precursor ion, 744.6 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.36 a) MS scan at 34.3 min showing one of the most intense 135
precursor ion, 759.5 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.37 a) MS scan at 43.2 min showing one of the most intense 136
precursor ion, 717.6 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
xvi
subunit.
Figure 3.38 a) MS scan at 30.7 min showing one of the most intense 137
precursor ion, 716.5 m/z which was subjected to MS/MS
scan; b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.39 a) MS scan at 54.6 min showing one most intense 138
precursor ion, 1086.6 m/z was subjected to MS/MS scan;
b) MS/MS spectrum; c) amino acid sequence derived
from MS/MS spectra which belong to ATP synthase beta
subunit.
Figure 3.40 The 36 medium intense common protein spots with 141
significant Mowse score (circled) detected in the 2-DE gel
of young and mature leaf extracts of P. sarmentosum
from seven geographical areas. Spot # 34, 41, 46, 47, 50,
51, 52, 54, 55, 62 and 64 were homology proteins
with Mowse score below 44.
Figure 3.41 The 14 lower intense common protein spots with 149
significant Mowse socre (circled) detected in the 2-DE gel
of young and mature leaf extracts of P. sarmentosum
from seven geographical areas. Spot # 66, 71, 77, 78, 81
and 83 were homology proteins with Mowse score below 44.
Figure 3.42 Bar chart of Differential expression of common protein 158
spots from young leaf extracts of P. sarmentosum obtained
from different geographical areas.
Figure 3.43 Bar chart of differential expression of common protein 166
spots from the mature leaf extracts of P. sarmentosum
obtained from different geographical areas.
Figure 3.44 Immunoblot of peroxiredoxin. The bands indicated the 188
presence of peroxiredoxin in the leaf extracts of
P. sarmentosum and accuracy of LCMS/MS analysis.
M: Marker; L1: young leaf extracts of P. sarmentosum;
L2: mature leaf extracts of P. sarmentosum.
Figure B1 Sequence alignment of ribulose-1, 5-bisphosphate 225
carboxylase/oxygenase large subunit
Figure B2 Sequence alignment of putative NAD(P)H oxidoreductase 229
isoflavone reductase (spot # 119, 123, 124), isoflavone
xvii
reductase-like protein 6 (spot # 125) and phenylcoumaran
benzylic ether reductase like protein (spot # 121 ).
Figure B3 Sequence alignment of disease resistance protein RPS4 230
(spot # 114 ) and putative disease resistance gene protein
(spot # 155 )
Figure B4 Sequence alignment of actin and actin-like protein 231
(spot # 59, 60, 61).
Figure B5 Sequence alignment of transketolase precursor (spot # 15, 19), 231
transketolase chloroplastic (spot # 12), transketolase-like
protein (spot # 13) and transketolase C terminal like
(spot # 14)
Figure B6 Sequence alignment of vacuolar ATP synthase catalytic 233
subunit A (spot # 17), vacuolar proton-ATPase subunit A
(spot # 19) and putative vacuolar ATP synthase subunit A
(spot # 20).
Figure B7 Sequence alignment of dnaK-type molecular chaperone 234
HSC 70-10 (spot # 194), dnaK-type molecular chaperone
precursor (spot # 20), dnakK-type molecular chaperone
hsp 70 (spot # 25), heat shock protein Hsp 70 (spot # 27)
and dnaK-type molecular chaperone HSC 70-9
(spot # 30, 31)
Figure B8 Sequence alignment of chaperonin groEL alpha chain 235
precursor (spot # 32), chaperonin groEL
(spot # 33, 34, 36, 46) and chaperonin 60 alpha
chain precursor (spot # 35, 38).
Figure B9 Sequence alignment of chaperonin 21 (spot # 190) 236
and groES-like (spot # 192)
Figure B10 Sequence alignment of translation elongation factor 237
EF-Tu precursor (spot # 67) and putative chloroplast
translation elongation factor EF-Tu (spot # 68, 166).
Figure B11 Sequence alignment of 3-phosphoglycerate kinase 237
(spot # 82), phosphoglycerate kinase chloroplast putative
(spot # 76), chloroplast phosphoglycerate kinase
(spot # 74, 78, 79, 80, 165), and phosphoglycerate
kinase precursor chloroplast (spot # 77)
xviii
Figure B12 Sequence alignment of peroxidase 1 (spot # 115, 116, 117), 238
L-ascorbate peroxidase 1b (spot # 163) and peroxidase
neutral (spot # 44).
Figure B13 Sequence alignment of phosphoribulose (spot # 84, 85) 239
and phosphoribulose precursor (spot # 87)
Figure B14 Sequence alignment of sedoheptulose-bisphosphatase 239
precursor (spot # 90), OSJNBa0042F21.B protein
(sedoheptulose-1, 7-bisphosphate) (spot # 91)
Figure B15 Sequence alignment of chloroplast malate dehydrogenase 240
(spot # 57, 86), cytosol malate dehydrogenase
(spot # 94, 97, 104, 107) and mitochondrial malate
dehydrogenase (spot # 114).
Figure B16 Sequence alignment of fructose-bisphosphate aldolase 241
precursor chloroplast (spot # 95, 97, 100, 105, 106, 108),
probable fructose-bisphosphate aldolase precursor
(spot #104, 107, 110, 113) and fructose-bisphosphate
aldolase chloroplast putative expressed (spot # 109).
Figure B17 Sequence alignment of ATP synthase gamma chain 241
(spot # 95, 96, 99) and H+- transporting two sector
ATPase gamma chain precursor (spot # 106)
Figure B18 Sequence alignment of oxygen-evolving enhancer 242
protein 1 chloroplast precursor (spot # 134, 135, 136),
photosystem II oxygen-evolving complex protein 1
precursor (spot # 137), probable photosytem II
oxygen-evolving complex protein 2 precursor
(spot # 197, 198, 201) and photosystem II
oxygen-evolving complex protein (spot # 209).
Figure B19 Sequence alignment of cytosol (spot # 167) and 243
chloroplast (spot # 173, 176) isoforms of triose
phosphate isomerase.
Figure B20 Sequence alignment of chlorophyll a-b binding 243
protein precursor (spot # 172) and chlorophyll
a/b-binding protein 2 precursor (spot # 186, 187)
Figure B21 Sequence alignment of 2-Cys peroxiredoxin 243
precursor (spot # 210, 212) and peroxiredoxin (spot # 228)
Figure B22 Sequence alignment of cysteine synthase (spot # 102, 127) 244
xix
LIST OF ABBREVIATIONS
2-DE : Two-dimensional gel electrophoresis
ACN : Acetonitrile
AEBSF : 4-(2-Aminoethyl) benzenesulfonyl fluoride
APS : Ammonium persulfate
BSA : Bovine serum albumin
CHAPS : 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate
CID : Collision induced dissociation
DTT : 1,4 – Dithiothreitol
ESI : Electrospray ionization
IEF : Isoelectric focusing
IPG : Immobilized pH gradient
kDa : kilo Dalton
LC-MS/MS : Liquid chromatography tandem mass spectrometry
MS/MS : Tandem mass spectrometry
MS : Mass spectrometry
m/z : Mass to charge ratio
NaCl : Sodium chloride
Na2HPO4.2H20 : Disodium hydrogen phosphate dihydrate
NaH2PO4.2H20 : Sodium dihydrogen phosphate dihydrate
NH4HCO3 : Ammonium bicarbonate
PAGE : Polyacrylamide gel electrophoresis
PBS : Phosphate buffered saline
xx
pI : Isoelectric point
RP-HPLC : Reverse-phase high performance liquid chromatography
SDS : Sodium dodecyl sulfate
TCA : Trichloroacetic acid
TEMED : N, N, N’ N’ - tetramethylethylenediamine
TLB : Thiourea lysis buffer
TRIS : Tris buffer
Tris : Tris(hydroxymethyl)aminomethane
xxi
LIST OF APPENDICES
Page
Appendix A: List of Chemicals 222
Appendix B: Sequence alignment of amino acid sequences that were 225
identified by LCMS/MS
Appendix C: Images of differential expression of common protein spots of 245
young leaf extracts from P. sarmentosum obtained from
different geographical areas
Appendix D: Images of differential expression of common protein spots of 251
young leaf extracts from P. sarmentosum obtained from
different geographical areas
xxii
PENYELIDIKAN ESTRAKAN PROTEIN DARIPADA DAUN PIPER
SARMENTOSUM
ABSTRAK
Suatu kajian proteomik berdasarkan penggunaan gel telah berjaya diusahakan ke
atas tisu daun P. sarmensotum. Suatu kaedah pengekstrakan protein yang dikenali
sebagai pengesktrakan kesemua protein diikuti dengan pengendapan trikloroacetik asid
(TCA)/ aseton telah digunakan dan telah dibuktikan menghasilkan ekstrak tisu daun
yang berkualiti tinggi dan serasi dengan pemisahan dua-dimensi elektroforesis gel. Profil
protein tisu daun P. sarmenstosum telah menghasilkan sejumlah 242 tompok protein dan
seterusnya 283 protein telah dikenalpasti dengan menggunakan analisa LCMS/MS.
Kebanyakan protein ini adalah terlibat dalam metabolisme dan protein-protein yang
berkaitan dengan destinasi dan simpanan protein, tenaga dan pengangkutan, pertahanan
dan tekanan. Dalam analisis komparatif (perbandingan) proteomic ke atas daun muda
dan dewasa yang diperolehi dari tujuh tempat yang berbeza, 84 tompok protein telah
dikesan dalam semua duan ekstrak samada daripada daun muda, daun dewasa mahupun
kawasan geografi. Daripada 84 tompok protein, 21 tompok protein masing-masing
daripada daun muda dan dewasa telah dikenalpasti sebagai protein ekspresi berbeza di
mana perbezaan antara keamatan tompok tertinggi dan tompok terendah dari tujuh
tempat yang berlainan adalah sebanyak 2 kali ganda. Tambahan lagi, 4 tompok protein
telah dikesan sebagai tompok unik dan 21 tompok protein dikesan sebagai tompok tak
teratur. Secara keseluruhannya, pemetaan proteome tisu daun P. sarmentosum telah
xxiii
berjaya mendedahkan komposisi protein dalam tisu daun P. sarmentosum, suatu
tanaman herbal yang nilai ubatannya terkandung dalam daun.
xxiv
ANALYSIS OF PROTEINS EXTRACTED FROM THE LEAVES OF PIPER
SARMENTOSUM
ABSTRACT
A gel based proteomic method has been developed for the leaf tissues of P.
sarmentosum. A suitable protein extraction method termed as total protein extraction
followed by trichloroacetic acid (TCA)/acetone precipitation was applied and has been
shown to produce good leaf tissue extract that was compatible with 2-DE separation.
The protein profiling of leaf tissues of P. sarmentosum showed a total of 242 protein
spots and from which 283 proteins were identified by LCMS/MS analysis. Most of these
identified proteins were involved in metabolism (including photosynthesis), protein
destination and storage, energy and transport, and defense and stress related protein. In
the comparative proteomic analysis on young and mature leaf tissues of P. sarmentosum
obtained from seven geographical areas, 84 protein spots were found common in all the
leaf extracts of P. sarmentosum regardless of young or mature leaf or geographical areas.
Among these 84 common protein spots, 21 protein spots respectively in the young and
mature leaves of P. sarmentosum were detected as differentially expressed proteins with
a 2-fold change in intensity between the highest intense spot and the least intense spot
when comparing between the seven geographical areas. In addition, 4 protein spots were
detected as unique spots and 21 protein spots were detected as irregular protein spots.
The proteome of leaf tissues of P. sarmentosum has been successfully mapped which
revealed the protein composition of the leaf tissue of P. sarmentosum, a traditionally
known leaf-based medicinal plant.
1
CHAPTER 1
INTRODUCTION
1.1 Traditional Medicinal Plants
A medicinal plant or herb is defined as a plant or plant part valued for its
medicinal, savory, or aromatic qualities (Kowalchik et al., 1998). Medicinal plants are
widely used in prevention and treatment of disease. They are also used as an ingredient
in health care supplements. The usage of the medicinal plants is normally based on
traditional knowledge, where most of them have not been scientifically proven. There
are a few drawbacks in the usage of medicinal plants; failure of the treatment includes
mis-usage, and its potential side effects resulted in the therapeutic usage being pressured
by World Health Organization (Caniato and Puricelli, 2003). However, 70-80% of world
population still rely on traditional herbal medicine as a primary source of disease
treatment (Caniato and Puricelli, 2003; Hamilton, 2004).
The demand of herbal medicine is still growing continually due to the
exponential increase in human population, less side effects and the low cost of
traditional medicine (Hamilton, 2004; Patwardhan et al., 2005). This phenomenon is
noted in the significant increase of the market for Ayuvedic and Chinese medicines
(Patwardhan et al., 2005). Today, more than 35, 000 herbal plant species in the world
are utilized appropriately in various parts of the world (Philip et al., 2009).
The systematic studies on medicinal plant are significantly increased recently,
where medicines derived from plants for the treatment of different diseases such as
cancer, microbial, fungal and viral infection has been developed (Hamilton, 2004; Philip
2
et al., 2009). Moreover, the need of traditional medicines as an alternative therapy for
drug resistance strain of microbial and fungal has been documented (Najib Nik A.
Rahman et al., 1999; Patwardhan et al., 2005). Many pharmaceutical companies have
started to focus on natural product chemistry for discovery and development of natural
product drug. As a result, more than 1500 medicine plants were developed and
processed into various forms of botanical products such as dietary supplements and
herbal teas (Patwardhan et al., 2005).
1.2 Piper
Genus Piper, under the family Piperaceae consists of more than 700 species
habitually distributed in tropical and subtropical regions of the world (Rukachaisirikul et
al., 2004). The Piper species include Piper nigrum, Piper longum L., Piper ribesoides,
Piper amalago, Piper brachystachyum, Piper sarmentosum and more (Parmar et al.,
1996; Scott et al., 2007). The genus Piper shares similar characteristics. They are erect,
creeping and small terrestrial tree chiefly possessing flower and fruit. The leaves, fruits,
stems and roots of the Piper species are commercially, economically and medicinally
valuable in the worldwide market (Scott et al., 2007).
The value of the fruit of P. nigrum is economically high, where products of white
and black pepper are used as spices in many tropical countries (Parmar et al., 1996). In
the regions of Latin America, West Indies and India, the genus Piper plant is well
known for their medicinal properties. Piper hispidum and Piper aduncum were used by
Jamaican to alleviate stomach aches. In the Ayuvedic medicine, Piper sylvaticum is an
excellent antidote for snakebite. Extensive phytochemical studies have revealed the
presence of various class of physiologically active compound such as lignans, steroids,
3
alkaloids, amides, pyrones, flavones, flavanones, in Piper species (Rukachaisirikul et al.,
2004; Scott et al., 2008). These physiologically active compounds were proven to be
important in plant chemical defense (de Morais et al., 2007). A number of species of
Piper contain piperamides, potential botanical insecticides that is used as pest controls to
prevent the damage of stored crops (Scott et al., 2008).
1.3 Piper sarmentosum
Piper sarmentosum, locally known as kadok, is mainly found in Northeast India,
South China, Indonesia, Thailand, and Malaysia (Rukachaisirikul et al., 2004). It is a
very popular and widely used herb in Malaysia as food flavouring agent and traditional
medicines (Nirwana et al., 2009). P. sarmentosum is used traditionally for treatment of
fever, coughs, flu, diabetes, hypertension rheumatism and joint aches. The plant appears
as glabrous, creeping, terrestrial herbs with about 20 cm to 50 cm tall (Rukachaisirikul et
al., 2004). Its flowers are either bisexual or unisexual. Its fruits are small and dry with
several rounded bulges. P. sarmentosum has a characteristic of pungent odour.
Studies have shown that P. sarmentosum contains antioxidant property
(Subramaniam et al., 2003). A naturally occurring antioxidant, superoxide scavenger,
Naringenin was isolated from the methanolic leave extracts of P. sarmentosum. It is
believed that frequently consuming the fruits or leaves of P. sarmentosum could
scavenge access free-radicals and superoxide anion (O2-) to prevent certain conditions
such as DNA damage, cardiovascular disease, stroke, hypertension and cancer
(Subramaniam et al., 2003). The positive effect of P. sarmentosum in treating caecal
amoebiasis in mice has also been reported (Sawangjaroen et al., 2004). Two amides
isolated from P. sarmentosum, namely sarmentine and 1-piperettyl pyrrolidine possess
4
antituberculosis and antiplasmodial activities (Rukachaisirikul et al., 2004). Pellitorine,
guineensine, brachyamide, sarmentosine and 1-(3, 4-methylenedioxyphenyl)-1E-
tetradecene extracted from the fruits of P. sarmentosum were reported to contain
antituberculosis activities (Rukachaisirikul et al., 2004). According to Peungvicha et al.
(1998), the water extract of P. sarmentosum has been tested to display hypoglycemic
effect on the streptozaotocin-diabetic rats.
1.4 Protein, polypeptide, peptide and amino acid
Protein is a polymeric compound composed of a set of monomers called amino
acid (Matthew et al., 2000). An amino acid contains a central carbon atom attached to an
amino group, a carboxylate group, a hydrogen atom and a side chain R. There are 20
amino acids found in human biological system, namely alanine (A), arginine(R),
asparagine(N), aspartic acid(D), cysteine(C), glutamic acid(E), glutamine(Q), glycine(G),
histidine(H), leucine(L), isoleucine(I), lysine(K), methionine(M), proline(P),
phenylalanine(F), serine(S), threonine(T), tryptophan(W), tyrosine(Y), and valine(V)
(Matthew et al., 2000; McKee, 2008).
The structure of amino acid enables it to behave as acid and base simultaneously
by donating a proton from its carboxylate group and accepting a proton to its amino
group at pH of 7. This phenomenon is called amphoteric. A condition with the presence
of both positive and negative charges on an amino acid is termed zwitterionic form. The
classes of amino acid are determined according to the solubility of the amino acid
(McKee and McKee, 2008).
Based on the amino acid solubility, the groups of amino acids were classified
into four groups: nonpolar, polar, acidic and basic. The side chain of nonpolar amino
5
acid mainly constitute of either aliphatic or aromatic hydrocarbon R group. The weakly
interaction of nonpolar amino acid with water is required to maintain three dimensional
structure of protein. Glycine, alanine, valine, leucine, isoleucine, proline, phenyalanine,
tryptophan, cystein and methionine are categorized as nonpolar amino acids. Polar
amino acid is referred as water loving amino acid. The hydroxyl group within the amino
acid enables the interaction between amino acid and water to take place through the
linkage of hydrogen bond. Serine, threonine, tyrosine, asparagine, and glutamine are
grouped as polar amino acids. Acidic amino acids carry negative charge on its side chain
at pH7 (Matthew et al., 2000). There are only two amino acids in acidic form: aspartic
acid and glutamic acid. In contrary, basic amino acids carry positive charge on its side
chain at pH 7. The group of basic amino acid includes histidine, lysine and arginine.
Amino acids linked together covalently to form peptide by removing a water
molecule between carboxyl group from one amino acid and amino group from other
amino acid (Matthew et al., 2000). The bond formed is termed as peptide bond. Amino
acid residues combined into a long chain is called polypeptide. Protein has one or more
polypeptide fold into three-dimensional structure to perform biological function. Post-
translation modifications, such as phosphorylation, glycosylation are common in protein.
These modifications play an important role in explicating the biological functions of
proteins (Seo and Lee, 2003). Proteins that are involved in a variety of biological
functions in living organisms, including catalyst, provide structural support, help in
transportation and movement, regulate stress response and cellular mechanism pathway,
storage of essential nutrient, and have protective function against mechanical and
chemical injury.
6
Proteins have four levels of structures: primary, secondary, tertiary and
quaternary structure (Branden and Tooze, 1999). Protein primary structure is described
as a polypeptide chain with defined amino acid sequence. Protein secondary structure is
the local arrangement of the backbone of a linear polypeptide to form a repeating
structure such as α-helix and -sheet structure. Extensive folding structure in a linear
polypeptide to form three-dimensional shape is referred as protein tertiary structure. A
protein quaternary structure is formed by two or more folded polypeptide chain to
achieve highest complexity. Depending on the types of amino acid that made up the
protein, protein will carry certain chemical property resulted from the synergistic
interaction of its amino acids component.
1.5 Proteomics
Proteome is depicted as entire protein complement of a particular biological
system (Cagney et al., 2003). Consequently, proteomics is related with systematic
analysis of protein population synthesized in cells (Boguski and Mclntosh, 2003;
Cagney et al., 2003). The completion of genome sequencing projects had urged the rapid
progress of proteomics to evaluate the genome expression after mRNA editing,
alternative splicing and post-translational modification. The emergence of proteomics is
greatly boosted by the emerging of new technologies such as the sensitive and high
throughput of mass spectrometry and high resolution of two-dimensional gel
electrophoresis (2-DE), which are the standard procedure for quantitative proteome
analysis (Boguski and Mclntosh, 2003).
7
The proteome is a challenging, complex and dynamic system influenced by
diverse physiological and pathological factor (Gromov and Celis, 2000). The
transcripted and translated genome varies in different type of cells. Usually only low
quantity of genome is transcripted and translated within a cell. Therefore, after
undergoing mRNA editing, alternative splicing, and post-translational modification, the
proteome variability is greatly different from cell to cell, and eventually species to
species (Gromov and Celis, 2000). The proteomics reveal the expression of gene
products, characterize protein composition, functions, activities, interactions and
distribution at different cellular stage (Gromov and Celis, 2000; Ong and Mann, 2005).
However, about 80-90% of total protein is monopolized by housekeeping protein with
the different expression patterns in majority of cells (Gromov and Celis, 2000).
1.6 Plant Proteomics
Plant proteomics which started in early 1980s, has progressed rapidly over the
few years with the completion of first plant genome sequence of Arabidopsis thaliana
(thale cress) in year 2000 (Canovas et al., 2004). Arabidopsis is a premier dicot model
species composed of five chromosomes with a total size of approximately 135 Megabase
(MB) (Rossignol et al., 2006). Small genome of Arabidopsis with a relative short life
cycle has made the species a popular tool in molecular biology studies such as flower
development. Plant proteomics is still in the beginning stage. Proteomic study is mainly
depending on availability of database. The slow growth in plant proteomics is due to the
scarcity of plant protein database (Nam et al., 2003; Katam et al., 2009).
Most proteomic studies are focus on Arabidopsis and Oryza sativa (rice). This is
mainly due to the completion of the genome sequence of these plant species, which
8
resulted in the availability of the protein databases in the public domain that facilitate the
process of protein identification using MS data. In the past few years, the complete
genome sequence of the grape, Populus trichocarpa (black cottonwood) and
Physcomitrella patens have been published (Quirino et al., 2010), which enhance the
progress of the plant proteomic research. Recently, the plant proteomics were further
extended to plants in which the genome sequence is available in significant number in
the forms of genomic DNA and Expressed Sequence Tag (EST) sequences. These
include Medicago truncatula (legume barrel medic) along with Solanum lycopersicum
(tomato), Zea mays (maize), Solanum tubersum (potato), sorghum and soybean, Quercus
ilex (holm oak), Spartium junceum (Spanish broom), Panax ginseng (ginseng), Pisum
sativum (pea), Pinus radiate (Radiate pine), Pinus halepensis (Aleppo pine), Ananas
comosus (pineapple) Phoenix dactylifera (Date palm) and, Embothrium coccineum
(Notro) (Jorrin et al., 2007; Jorrin-Novo et al., 2009). The scarcity database entries of
other plants group is compensated for by protein identification through de novo MS
sequencing and BLAST searches.
Since plant proteomics have generated a huge number of data, some of the
researchers have made an effort on the database organization. The examples of current
available database are Plant Proteome DataBase (PPDB), http://ppdb.tc.cornell.edu/
mainly for Arabidopsis and Z. mays; Soybean proteome Database,
http://proteome.dc.affrc.go.jp/Soybean/; The Plant Organelles Database 2 (PODB2),
http://podb.nibb.ac.jp./Organellome/; Protein Mass Spectra Extraction (ProMex),
http://promex.mpimp-golm.mpg.de/home.shtml; Rice Proteome Database,
http://gene64.dna.affrc.go.jp/RPD/, etc. The ProMex, is a MS/MS spectral database
9
derived from Arabidopsis, Chlamydomonas reinhardtii, Medicago truncatula, Solanum
lycopersicum, Solanum tubersum and other plants.
In proteomic, the plant tissue parts that had been analyzed include seeds, roots,
stems, leaves, pistils, xylem or phloem saps, pollens and whole seedlings (Jorrin et al.,
2007). Generally, the plant proteomic studies focus on protein profile of plant tissue
parts in order to examine the developmental changes of plant and influence of
environmental factors. The main focus of plant proteomic is on Arabidopsis, O. sativa
and Z. mays. Studies on the seed germination of Arabidopsis by 2-DE technique have
shown 1300 proteins in seeds, 74 of the presence of the proteins showed expression
changes during the germination process. A protein named gibberellins (GAs) was
reported to involve in the initial stage of germination (Gallardo et al. 2001; 2002). A
study on proteome of root, leaf, and seed tissues of O. sativa was carried out by using 2-
DE and LC-based separation methods (Koller et al., 2002). The authors reported that
only 7.5% of a total of 2528 proteins were expressed in all three plant tissues, showing
the differential expression of specific proteins in different parts of the plant. In 2001,
Porubleva et al. studied the maize leaf proteome where a total of 1100 proteins were
detected on 2-DE gels. The protein spots were then subjected to the MS analysis and
subsequently EST databases search. The outcome summarized that over 50% of proteins
cannot be identified from the database.
Studies on P. sativum and M. truncatula (Schiltz et al., 2004) for identification of
the nitrogen mobilization from leaves to seed filling in P. sativum resulted in
establishment of proteome reference maps of mature leaves and stems. The result
showed the importance of Rubisco in nitrogen mobilization and 14-3-3 like protein as a
potential regulator during the massive remobilization of nitrogen. Study of proteome
10
patterns was performed in leaves, stem, roots, flowers, seed pods and cell suspension
cultures of model plant M. truncatula (Watson et al., 2003). Three hundred and four
proteins out of 551 proteins were identified using peptide mass fingerprinting through
matrix-assisted laser desorption ionization time-of-flight mass spectrometry. When the
expression levels of the identified proteins were compared to the mRNA levels through
EST counting, approximately 50% of the proteins can be associated with their
corresponding mRNA levels. A similar study on Cannabis sativa to compare the
proteome of leaves, flower and glands was carried out (Raharjo et al., 2004). In this
study, the authors found that less than half of proteins expressed in flowers and glands as
compared to the leaves.
Recently, the proteomic of specific protein function in plant subcellular
compartments of chloroplast and mitochondria was investigated (Thiellement et al.,
2002; Jorrin et al., 2007). By sub-fractionating into hydrophilic and hydrophobic
condition for the better understanding of proteins functional roles, 81 proteins were
identified from the mitochondria of Arabidopsis (Millar et al., 2001). A series of study
on the chloroplast proteins of Arabidopsis and spinach was carried out on the envelope
and thylakoids (Rossignol et al., 2006), lumenal (Schubert et al., 2002), ribosomal
(Yamaguchi and Subramanian, 2000) and stromal proteins (Ytterberg et al., 2006). In
addition, Peltier et al. (2001) have identified a chloroplastic protease complex from
Arabidopsis. Recently, the development of proteome on the cell walls, membranes,
nucleus and vacuoles of plant cells were sequentially introduced to reveal the knowledge
of protein location and function in order to understand the plant biological processes
including plant development and growth, seed germination, signal transduction and the
responses of plants to symbionts, biotic and abiotic stresses (Rossignol et al., 2006;
11
Jorrin et al., 2007). Since the last decade, many reports have shown the potential of plant
proteins in inhibition of fungal growth. In 2000, Giudici et al. had successfully isolated a
16 kDa protein named SAP 16 from Helianthus annus. SAP 16 functions as trypsin
inhibitor that restricts the ascospore germination of Sclerotinia sclerotiorum and the
growth of mycelia. Other antifungal proteins (AFPs) reported were glucanases (Ng and
Ye, 2003), chitinases (Kitajima et al., 2010), thaumatin-like proteins (Wang and Ng,
2002) and several types of basic cysteine rich proteins and proteinase inhibitors (Joshi et
al., 1998) that were found in various plant species. Recently, the studies on the
proteomic of subcellular plant has been shifted from the technique 2-DE to a non-gel
based approaches which have been used in Arabidopsis species. In another study, Morel
et al. (2006) used multidimensional protein identification technology (MudPIT) to study
the proteome of detergent resistant membrane from plant tobacco. MudPIT is a
technique introduced by Yates laboratory for protein separation and identification using
two-dimensional liquid chromatography (2-D LC) coupled with a mass spectrometer
(Link et al., 1999; Lohrig and Wolters, 2009).
Malaysia is a country gifted with valuable types of plants that had been used
traditionally for their medicinal values. Although many research have been carried out
on these plants, most of them concentrated on the small molecule compounds instead of
the valuable macromolecule compounds of these plants.
1.7 Sample preparation
Sample preparation is the key step to determine the success of the experiment. In
plant proteomics, protein extraction is a great challenge because plant tissues normally
contain low amount of protein and high quantity of protease, phenolic compounds,
12
secondary metabolite, lipids and other non-protein compounds (Cristina-Maria and Katja,
2006; Isaacson et al., 2006; Weiss and Gorg, 2007). Thus, choosing an appropriate
method is critical in producing a high quality and reproducible result. Several protein
extraction methods have been developed due to the varied nature of plant tissue. Three
critical aspects that need to be considered in plant protein extraction namely (1) tissue
disruption, (2) removal of interfering compounds and (3) protein solubilization (Wang et
al., 2008):
(1) Plant cells are surrounded by a thick complex cell walls polysaccharide, cellulose
containing hemicellulose and pectin which are difficult to disrupt (Shewry and Fido,
1996; Jimenez et al., 2001; Isaacson et al., 2006). Plant cell wall provides rigidity
strength and protective structure against mechanical stress to the plant cells. As a
consequence, a proper tissue disruption step is required to completely destroy the cell
wall. The most widely used technique to break down the plant cell walls is by freezing
the plant tissues with liquid nitrogen and then grinding the plant sample using mortal
and pestle (Wittmann-Liebold, 2006; Wang et al., 2006; 2008). Liquid nitrogen, a
cryogenic liquid, possesses boiling point as low as -196 C which can cause rapid
freezing on the plant tissue (Chawla, 2002). The employment of liquid nitrogen with
cryogenic property gives the extra advantage in prevention of protein degradation and
proteolysis. Quartz sand can be added to assist the grinding process (Wang et al., 2008).
(2) Plant tissues have comparatively low protein content while high contaminants of
secondary metabolites accumulate inside the vacuoles of plant cells (Cristina-Maria and
Katja, 2006; Isaacson et al., 2006; Weiss and Gorg, 2007). A vacuole contains tonoplast
that enclosed the inorganic and organic molecules such as polysaccharides, proteases,
13
toxic compounds, phenolic compounds, alkaloids, flavonoid pigments, tannins, lignins,
etc (Taiz, 1992; Weiss and Gorg, 2007). The presence of all these interfering compounds
in the vacuoles varies with the development stage of a plant and varies from species to
species. Generally, these compounds were found rich in adult plant tissues than in young
tissues (Wang et al., 2008). The removal of secondary metabolites in extraction step is
absolutely important to obtain a high quality 2-DE result. The existence of secondary
metabolites such as pigments, lipid and polysaccharides will interfere the performance
of 2-DE, especially the phenolics. Phenolics contain aromatic ring with one or more
hydroxyl groups, they are mainly derived from the pentose phosphate, shikimate and
phenylpropanoid pathways (Balasundram et al., 2005). Phenolics which form a complex
with the protein irreversibly can cause the 2-DE gels streaking and artifactual spots (Xu
et al., 2008). The application of the 10% (w/v) trichloroacetic acid (TCA)/acetone
precipitation on plant extract is an effective way to remove the secondary metabolites,
either before or after the protein extraction. The resulting pellet from the TCA/acetone
precipitation should be white or light yellowish in color. The addition of polypyrrolidone
(PVPP) in extraction buffer can precipitate the phenolic compounds through the
formation of PVPP-phenolic complex and the elimination of the complex can easily be
done through centrifugation (Wang et al., 2008).
(3) Solubility of protein in plants varied from species to species, depending on the
nature of the plant tissue. The protein solubilization normally includes the breaking
down of the macromolecular interactions in order to produce individual polypeptide
chains via disruption of disulfide bonds and non-covalent interactions, for example ionic
bonds, hydrogen bonds and hydrophobic interactions inside the proteins and non-
covalent interactions between proteins and non-proteinaceous compounds such as lipids,
14
carbohydrate and nucleic acids (Rabilloud, 2002). The ideal protein extraction condition
is to maintain all the extracted polypeptides in the structure similar to their exact living
state. Thus, artifact modifications of the polypeptides have to be prevented by the
appliance of low temperature condition and protease inhibitor cocktail to the plant
tissues. This step is important to inactivate all the proteases that are involved in protein
modification (Lopez, 2007). Protease inhibitor and reducing agent can also be used to
improve protein solubilization (Wang et al., 2008).
1.8 Protein Extraction buffer
In plant proteomics, a strong extraction buffer is required to extract total protein
from the tissues. The most common extraction buffer used in the plant proteomics,
especially for extraction of leave proteins always contains chaotrope, denaturant,
detergent and reductant (Cristina-Maria and Katja, 2006; Lopez, 2007).
Urea is a neutral chaotrope which acts as a denaturant to solubilize and unfold
proteins via breaking their hydrogen bonds into a random conformation in which all
ionizable amino acid side chains are exposed to buffer solution (Monribot and Boucherie,
2000; Rabilloud, 2002). Urea normally functions at concentration as high as 7 M in the
presence of thiourea for further solubilization improvement, this is especially true for the
hydrophobic proteins (Rabilloud, 2002). At high concentration, urea in the structure of
organized channels can directly bind to linear alkyl chains molecules to form a complex
called inclusion compound. These inclusion compounds are less soluble and the
precipitation is often induced in the presence of nonionic or zwitterionic detergents with
basic linear alkyl chains structure. Therefore, the choice of the detergents is limited to
non-linear alkyl tails, for example: Tritons, Nonidet P40, 3-[(3-cholamidopropyl)
15
dimethylammonio]-1 propanesulfonate (CHAPS) and octyl glucoside. Urea can easily
break down to form isocyanate through a process called protein carbamylation. For this
reason, the use of protein extraction buffer containing urea should be completely
avoided at temperature above 37C in order to reduce the formation of artifactual spots
in 2-DE gels (Rabilloud, 2002). Other precautions are to avoid using of low purity urea
or adding of cyanate scavenger into a urea solution.
Nonionic or zwitterionic detergents are required to solubilize protein completely
while preventing protein aggregation through hydrophobic interactions (Lopez, 2007). In
several studies, ionic detergent such as sodium dodecyl sulfate (SDS) was added in
protein extraction. The presence of SDS as an anionic surfactant assists the protein
solubilization by disrupting non-covalent interactions and subsequently produces
electrostatic repulsion in order to separate polypeptides (Monribot and Boucherie, 2000;
Rabilloud, 2002). However, the utilization of SDS is incompatible with IEF due to its
ionic nature (Mechin et al., 2007). Therefore, SDS is always used in a diluted form in
extraction solution with high concentrations of urea and nonionic or zwitterionic
detergents (Rabilloud, 2002). Only low amount of SDS at a concentration lower than
0.25% or at a ratio at least 8:1 (detergent to SDS) is tolerated with the use of IEF
(Righetti et al., 2001).
Reducing agents are used to reduce protein by breaking its disulfide bonds
during the protein extraction process (McGettrick and Worrall, 2003). Dithiothreitol
(DTT) and 2-mercaptoethanol are universal reductants being used (Rabilloud, 2002).
However, the usage of DTT is preferable due to formation of artifacts by 2-
16
mercaptoethanol. Moreover, high concentration of 2-mercaptoethanol is needed
compared to DTT, which DTT only used at concentration ranging from 20 to 100 mM.
1.9 Protein Precipitation and Purification
The application of the 10% (w/v) trichloroacetic acid (TCA)/acetone
precipitation can be performed either before the protein extraction or after the protein
extraction (Wang et al., 2008). In the former usage, plant tissue powder is directly
subjected to TCA/acetone precipitation before the protein extraction. In contrary in the
latter usage, the tissue powder is extracted with aqueous buffer and the extract is
subjected to TCA/acetone precipitation. The main purpose for TCA/acetone
precipitation is to purify and concentrate the protein before running the 2-DE. This
protein purification and precipitation technique, primarily described by Damerval et al.
(1986) is more effective than the purification using trichloroacetic acid or acetone alone.
10% TCA can effectively remove majority of secondary metabolites such as lipid,
pigments and phenolic compounds under acidic and hydrophobic conditions (Wang et
al., 2008). Moreover, the use of TCA in acetone can inhibit the activities of proteases,
phenoloxidases and peroxidases by preventing proteolytic degradation and thus, loss of
high molecular weight proteins (Mechin et al., 2007). The lyophilized pellet from
TCA/acetone precipitation cannot be over-dried to ensure the efficiency of
resolubilization.
1.10 Protein Assay
The protein assay used in this study is a colorimetric assay for protein
quantitation based on the Lowry method (Lowry et al., 1951). The assay employs two
17
different reactions. First, the protein reacts with copper ion to form a reduced copper in
alkaline solutions. Second, the copper-amide bond complex from first reaction reduces
Folin-Ciocalteu reagent (phosphomolybdate and phosphotungstate) (Olson and
Markwell, 2007). The protein amount is spectrophotometrically detected via the
formation of reduced Folin-Ciocalteu reagent, which is blue in color with the absorption
in the range of 500 to 750 nm wavelength. The Lowry assay is sensitive, however, the
sensitivity of Lowry assay is reduced by some interferent such as chlorophyll, detergents,
carbohydrates, glycerol, Tricine, EDTA, Tris, potassium compounds, sulfhydryl
compounds and disulfide compounds (Berges et al., 1993; Olson and Markwell, 2007).
Several modified Lowry assay as well as commercialized ones have been developed to
eliminate the interferents. The commercialized protein assay has been modified to be
reducing agent and detergent-compatible.
1.11 Two-Dimensional Gel Electrophoresis
Two-dimensional gel electrophoresis is a powerful technique used to separate
and analyze proteins according to isoelectric point (pI) in the first dimension and
molecular weight in the second dimension (Stochaj et al., 2002; Xu et al., 2008). The
use of two-dimensional gel electrophoresis in proteome studies is first described by O’
Farrell and J. Klose in 1975 (Stochaj et al., 2002). However, the original technique is not
commonly used due to handling problem and the complexity of result.
Recently, the two-dimensional gel electrophoresis has been successfully
improved in the aspects of reproducibility, resolution, and separation of highly acidic
and basic proteins (Weiss and Gorg, 2007). The new immobilized pH gradients were
introduced by Gorg and colleagues to replace the old carrier ampholyte-generate pH
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gradients. Moreover, the availability of second generation software and powerful
computer assisted software facilitate the complicated protein analysis process (Gygi et
al., 2000; Wittmann-Liebold, 2006). Two-dimensional gel electrophoresis is able to
separate amount of protein as low as 1 ng of protein content per spot, with detection of
more than 5000 proteins per gel by the combination use of highly sensitive protein
detection method (Weiss and Gorg, 2007). The deliverable result is presented as a map
that characterizes the protein profile of a biological system, which reveals the protein
expression level, isoforms or post-translation modification for a target protein (Barnouin,
2004).
1.11.1 Isoelectric focusing
Isoelectric focusing is the first dimension of two-dimensional gel electrophoresis.
It is carried out by using an immobilized pH gradients (IPG) strip (Stochaj et al., 2002).
The separation method of protein is based on their isoelectric point under an electric
field (Garfin, 2003). Isoelectric point of a protein is the pH of the local environment
when protein charge is zero (Rabilloud, 2002). The net charge of a protein is a total of its
positive and negative charges. It is a very environment dependent value. A protein
carries a negative charge when the environment pH is above its pI value, and conversely,
it carries a positive net charge when the surrounding pH is below its pI value (Lopez,
2007). Under a pH gradient, a positively charged protein migrates to the cathode with
decreasing charge density until its gain a zero net charge (Garfin, 2003; Barnouin, 2004).
The pI value is determined by the characteristic of side chains and prosthetic groups of
the protein; an acidic protein has a low pI value, while the basic protein has a high pI
value. The resolution of isoelectric focusing depends on the strength of electric field and
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the range of pH gradient. High voltage and narrow pH gradient will give high resolution
isoelectric focusing (Stochaj et al., 2002).
In 1982, Bjellqvist developed a new immobilized pH gradient for isoelectric
focusing instead of carrier ampholyte-generated pH gradient. The covalent fixed pH
gradient has resolved the problem of cathodic drift generated by carrier ampholyte-
generated pH gradient. In the method of immobilized pH gradient, Bjellqvist had
introduced the use of acrylamide buffer into polyacrylamide gel by copolymerize
acrylamide with bisacrylamide monomers. This is the present standard approach in
proteomics, which effectively improved the reproducibility, increase loading capacity
and broader pH range separation for protein. After isoelectric focusing, disulfide bridges
in protein are reduced and the sulfhydryl group is alkylated using dithiothreitol and
iodoacetamide respectively, before being subjected to the second dimension, which is
gel electrophoresis separation (Weiss and Gorg, 2007).
1.11.2 Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE)
Electrophoresis is a technique of separating charged particle such as proteins,
peptides and nucleic acids in an electric field (Eswara-Reddy and Jacobs, 1997). The
gels used in an electrophoresis usually can be made of a variety of sources including
starch, agarose, polyacrylamide and cellulose acetate.
Polyacrylamide gel is composed of nonionic polymerization of acrylamide and
crosslinking agent, N, N’-methylene-bis-acrylamide, the polymer is chemically stable
over a wide range of pH, temperature and ionic strength (Walker et al., 2000). The
concentration of acrylamide and cross-linker can be altered easily to produce different
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pore size of the gel, allowing proteins to separate in a particular range of molecular
weights (Stochaj et al., 2002).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a general technique
used to separate a mixture of protein based on their molecular weight. The presence of
sodium dodecyl sulfate in polyacrylamide gel acts as an anionic surfactant by
surrounding denatured protein in an approximate ratio 1.4:1 (sodium dodecyl sulfate:
protein) to form a net negative charged complex (Dunn and Bradd, 1993; Stochaj et al.,
2002; Lopez, 2007). In an applied electric field, the negative chargedly sodium dodecyl
sulfate-protein complex migrated towards the anode based on their molecular weights; a
low molecular weight protein moved faster than a high molecular weight protein
(Walker et al., 2000). In 2-D gel electrophoresis, a sodium dodecyl sulfate
polyacrylamide gel electrophoresis is based on Laemmli discontinuous Tris-chloride/
Tris-glycine system (Stochaj et al., 2002). However, no stacking gel is necessary due to
the presence of pre-separated step by isoelectric focusing. Protein resolution of two-
dimensional gel can be influenced by gel size. A larger gel is capable of detecting higher
number of protein resulting in a complex profile analysis. The appropriate acrylamide
concentration for two-dimensional gel electrophoresis ranges from 10-12.5% for
separation of protein size range of 10-200 kDa (Dunn and Bradd, 1993). Lower
percentages of acrylamide decrease the resolution of two-dimensional maps, while the
higher percentages complicate the later analysis.
1.12 Staining
There are diverse staining methods with different sensitivities available for
protein spots detection in two-dimensional gels, these staining methods include
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Coomassie Brilliant Blue, silver and fluorescence staining (Wirth and Romano, 1995;
Stochaj et al., 2002). A selected staining method in an analysis has to fulfill several
criteria including sensitive enough for low abundant protein detection, allow further
proteomic analysis and compatible with mass spectrometry. However, neither one of the
staining techniques is perfect. None of the techniques is able to stain all the present
proteins in a gel. Thus, different protein staining patterns may be obtained from different
staining techniques. Coomassie Brilliant Blue is the widely use, economical and simple
organic-based stain for post-electrophoretic protein detection (Weiss et al., 2009). The
narrow dynamic range of Coomassie Brilliant Blue dye only enables it to detect
approximately 0.5-2.0 μg of protein concentrations range (Stochaj et al., 2002).
Therefore, low abundance proteins below the sensitivity limit is precluded (Weiss et al.,
2009). Coomassive blue staining is an appropriate and reliable method for further
quantitative analysis coupled with computer software programs. Moreover, the dye can
be completely removed from the stained protein making it compatible with the
subsequent mass spectrometry analysis (Shevchenko et al., 2007).
1.12.1 Coomassie Brilliant Blue-R
Coomassie Brilliant Blue-R (reddish hue) is a non-polar, sulfated aromatic dye
that complexs with the basic amino acids, arginine, lysine, histidine as well as tyrosine
in order to visualize the presence of protein in a gel, with a limit of protein detection
about 30-100 ng (Wirth and Romano, 1995; Stochaj et al., 2002). Regressive staining
approach is used to destain Coomassie Brilliant Blue-R stained gel using a similar
solution as staining solution devoid of dye (Patton, 2002). The staining solution is a
mixture of dye with an aqueous solution containing methanol and acetic acid. During the
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destaining step, the stained gel background is gradually diminished, leaving the well
labeled protein band due to the higher affinity of proteins to the dye molecules than the
gel matrix.
1.12.2 Coomassie Brilliant Blue-G
Coomassie Brilliant Blue-G binds to the basic amino acids of protein. Unlike the
Coomassie Brilliant Blue-R, Coomassie Brilliant Blue-G contains colloidal dye particles,
as well as free dispersed dye in solution (Patton, 2002). During a staining equilibrium,
the low concentration of free dye preferentially penetrate and stain the protein, while the
colloidal dye particle of Coomassie Brilliant Blue-G (greenish hue) is incapable of gel
matrix penetration. Thus, a clear background can be obtained in a short period of
destaining step (Wirth and Romano, 1995; Weiss et al., 2009). The amount of protein
for detection range of Coomassie Brilliant Blue-G is approximately 8-50 ng, which is
more sensitive compared to Coomassie Brilliant Blue-R (Choi and Yoo, 2002; Stochaj et
al., 2002).
1.13 In-gel Digestion
In-gel digestion is a pre-step for mass spectrometric analysis. It is purposed to
extract, digest and degrade proteins into peptide form through an enzymatic cleavage
activity (Granvogl et al., 2007). The in-gel digestion method was initially established by
Rosenfeld et al. (1992) and several alterations have sequentially been proposed to assure
the high peptide yield and quality of mass spectrometric analysis (Granvogl et al., 2007).
Enzymatic cleavage with a specific protease results in a set of characteristic peptides
23
with diverse molecular masses. The molecular masses of peptides can be used to give
the identity of protein upon mass spectrometry.
Ammonium bicarbonate and Tris/hydrochloric acid are commonly used as
inorganic buffer salts in the process of in-gel digestion. Unlike the Tris/ hydrochloric
acid, ammonium bicarbonate is basic and therefore no pH adjustment is needed.
Moreover, ammonium bicarbonate can be completely removed and evaporated by
vacuum concentrator into ammonia gas, carbon dioxide and water. The continually
applied inorganic buffer salts and acetonitrile respectively in the washing and
dehydration steps efficiently destained the gel pieces by swelling and shrinking of the
gel pieces (Shevchenko et al., 2007). Finally, white dried gel pieces are obtained, which
was then saturated with a specific protease for protein digestion purpose. After an
incubation period, the peptide digestion product is extracted with solution for peptide
recovery (Kumarathasan et al., 2005).
1.13.1 Trypsin
Among the commercially available enzymes, the serine endopeptidase trypsin is
mostly used (Smith and Wheeler, 1996). Trypsin hydrolyse the polypeptide chain
specifically at arginine and lysine on the side of carboxyl terminal (Granvogl et al.,
2007). The hydrolysis rate is reduced if acidic or prolyl residues occur next to the
cleavage site. Typically, trypsin can function actively between pH 7 and 9 with an
optimum pH approximately at 8. At extremely acidic or basic condition, the activity of
trypsin can be reversible inactivated (Smith and Wheeler, 1996).
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1.14 Liquid Chromatography- Mass Spectrometry
The coupling of mass-spectrometry to liquid chromatography is a very sensitive
and reliable technique that can be applied to a wide range of biological samples. This
coupling technique progressed sluggishly in the early age. The employ of liquid
chromatography-mass spectrometry is limited due to the incompatibility of the early
mass spectrometric ion source with the liquid chromatographic system. In the 1980s,
Fenn developed an interface termed as electrospray ionization source to remove the
chromatographic mobile phase before further analysis by mass-spectrometry. This
development had carried a great impact on proteomic studies (Pitt, 2009).
Liquid chromatography is used to separate a complex protein mixture (Simpson,
2002). However, identity of an unknown analyte cannot be determined based on its
similarity to reference retention time alone (Ardrey, 2003).
Mass spectrometry is powerful technique for compound identification and
molecular structure study (Larsen and Roepstorff, 2002). The application of mass
spectrometry is incapable to obtain single mass spectrum for an analyte of the mixture.
The data for all compound ions is reported in a mass spectrum leads to complicated
analysis. Thus, coupling of high performance liquid chromatography with mass
spectrometry enables definitive identification through mass determination and the
quantitative analysis of the compound of interest.
1.14.1 Reverse-phased High Performance Liquid Chromatography (RP-HPLC)
The International Union of Pure and Applied Chemistry (IUPAC) defines
chromatography as “a physical method of separation in which the components to be
separated are distributed between two phases, one of which is stationary (the stationary
25
phase), while the other (the mobile phase) moves in a definite direction.” The mobile
phase for the liquid chromatography is a mixture of solvent which distributes on a
column (stationary phase) in a definite direction (Ardrey, 2003). High performance
liquid chromatography which carries out a reverse phase chromatographic separation
utilizes a polar mobile phase with a non-polar stationary phase (Simpson, 2002).
The elution time for an analyte to move out from the chromatographic column is
termed as retention time (tan). However, it is influenced by column length and mobile
phase flow rate. Thus, capacity factor (k’) is used to demonstrate the retention time of an
analyte and the unretained compounds (Simpson, 2002, Ardrey, 2003).
In proteomic studies, the high performance liquid chromatography separates
protein based on the interaction of the hydrophobic binding between amino acid side
chain of a protein and hydrophobic surface of chromatographic column (Simpson, 200).
Typically, a mixture of solvent is use in the high performance liquid chromatography
mobile phase. The initial condition of mobile phase always started with a highly aqueous
solvent followed by modification of subsequent mobile phase composition to produce a
gradient condition along the chromatographic separation process. Thus, a highly polar
protein is eluted earlier than the less polar protein. The high performance liquid
chromatography consists of five components: pump, injector, mobile phase, column and
detector (Ardrey, 2003). However, the separated samples are further analysed and then
detected in the mass spectrometry. Thus, the detector in the high performance liquid
chromatography is not being used.