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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.

iii

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

iv

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


3.1.1 Plant Cells Disruption Methods 48


3.1.2.1 Comparison of Extraction Methods Using SDS-PAGE 52

vii

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

viii

3.5 Unique Protein Spots 169

3.6 Irregular Protein Spot 173

3.7 Western Blotting 188

CHAPTER 4 – DISCUSSION 189


4.1.1 Plant Cell Disruption Methods 189


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

ix

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.


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.


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

xi

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.

xii

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.


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.


P. sarmentosum from Botanical Garden, Penang. b) 181


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




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.


P. sarmentosum from Johor. b) 206 protein spots were

detected in the 2-DE gel of the young leaves of

xiii

P. sarmentosum from Johor. c) Protein profiles of 2-DE

gel of the young leaves of P. sarmentosum from Johor.


young leaves of P. sarmentosum from Johor.


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.


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.


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.


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.


P. sarmentosum from Botanical Garden, Penang. b) 181

protein spots were detected in the 2-DE gel of the mature


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

xiv



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


Sarawak. d) 187 protein spots were detected in the


Sarawak.


P. sarmentosum from Johor. b) 148 protein spots were


P. sarmentosum from Johor. c) Protein profiles of 2-DE

gel of the mature leaves of P. sarmentosum from Johor.


mature leaves of P. sarmentosum from Johor.


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.


P. sarmentosum from Kedah. b) 169 protein spots were


P. sarmentosum from Kedah. c) Protein profiles of


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.

xv

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.





subunit.





subunit.





subunit.





subunit.





xvi

subunit.





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


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

http://ppdb.tc.cornell.edu/

http://proteome.dc.affrc.go.jp/Soybean/

http://podb.nibb.ac.jp./Organellome/

http://promex.mpimp-golm.mpg.de/home.shtml

http://gene64.dna.affrc.go.jp/RPD/

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

18

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

19

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

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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.

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