ISOLEVUGLANDIN-PROTEIN CROSS-LINKING - OhioLINK ...

PART I: ISOLEVUGLANDIN-PROTEIN CROSS-LINKING:

STRUCTURE AND MECHANISM

PART II: GENERATION AND CHARACTERIZATION OF A

MONOCLONAL ISOLEVUGLANDIN[4]-PROTEIN ADDUCT

ANTIBODY

by

WENZHAO BI

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis Advisor: Dr. Robert G. Salomon

Co-Advisor: Dr. John W. Crabb

Department of Chemistry

CASE WESTERN RESERVE UNIVERSITY

August 2014

CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

candidate for the Doctor of Philosophy degree *.

(signed) Dr. Michael G. Zagorski . (Chair of the committee)

Dr. James D. Burgess .

Dr. Rajesh Viswanathan .

Dr. John W. Crabb .

Dr. Robert G. Salomon .

(Date) 04 /28 /2014

*We also certify that written approval has been obtained for any proprietary materialcontained therein.

We hereby approve the dissertation of

Wenzhao Bi

This thesis is dedicated to my parents and my husband.

iii

TABLE OF CONTENTS

Table of Contents iv

List of Schemes vii

List of Tables x

List of Figures xi

Acknowledgements xvii

List of Abbreviations and Acronyms xix

Abstract xxiv

Part I: Isolevuglandin-protein Cross-linking: Structure and Mechanism

Part II: Generation and Characterization of a Monoclonal

Isolevuglandin[4]E2-protein Adduct Antibody

1. Introduction 1

1.1 Lipid Peroxidation 2

1.2 Enzymatic Pathways 2

1.3 The Free Radical Pathway 4

1.4 Biological Activities of Levuglandins and Isolevuglandins 7

1.5 References 22

2. Adduction and Cross-linking of Calpain-1 and GAPDH by Iso[4]LGE2

33

2.1 Background 34

2.2 Results and Discussion 38

2.3 Conclusions 58

iv

2.4 Experimental Procedures 60

2.5 References 71

3. Detection and Characterization of the Products from the Reaction of

Iso[4]LGE2 with Peptides: N-Acetyl-gly-lys-OMe and Amyloid

“EVHHQKL” 80

3.1 Background 81


3.3 Conclusions 108


3.5 References 114

4. Generation of IsoLG-pyrrole by the Reaction of IsoLG with N-Acetyl-

gly-lys-OMe in the Absence of O2 and the Oxidation of IsoLG-pyrrole

to Form Pyrrole-pyrrole Cross-links and an Adduct with N-

Acetylcysteine 120

3.1 Background 121


3.3 Conclusions 147


3.5 References 155

5. Generation of an Anti-iso[4]LGE2 Monoclonal Antibody 159

v

5.1 Background 160


5.3 Conclusions 169


5.5 References 185

Appendix 187

Bibliography 236

vi

LIST OF SCHEMES

Chapter 1

Scheme 1.1. Cyclooxygenase oxidation of AA generates PGs and LGs via

rearrangement of PGH2

3

Scheme 1.2 Schematic representation of rearrangement of PGH2 to

levuglandin.

4

Scheme 1.3 Generation of isolevuglandins and isolevuglandin-protein adducts

during free radical oxidation of AA-esters

5

Scheme 1.4 Formation of LG-protein adducts, protein-protein and DNA-

protein cross-links

6

Chapter 2

Scheme 2.1 Postulated structures of LG and iso[4]LG protein adducts and

cross-links.

35

Scheme 2.2 Synthesis of a fluorescently labeled iso[4]LGE2 derivative. 50

Scheme 2.3 AccQ·TagTM derivatization. 54

Scheme 2.4 Chemical structure of peptides aminal cross-link. The adduct

molecular weight was 604.3.

56

Chapter 3

Scheme 3.1 Schematic representation of pyrrole cross-linking structure in

previous study: adducts of PGJ2 reaction with GSH in HCA-7

81

vii

cells.


previous study: adducts of nucleosides and DNA formed by

reaction with levuglandin.

82


previous study: formation of bis(levuglandinyl) urea.

83


previous study: nucleophilic substitution of alkylpyroles in the

presence of oxygen.

83


previous studies: (A) Pyrrole autoxidation products in 2,5-

hexanedione-treated dipeptide. (B) Formation of thiol-pyrrole

conjugates.

84

Scheme 3.6 Schematic representation of formation of iso[4]levuglandin protein

adducts and cross-links.

85

Scheme 3.7 Possible structures for products of oxidized bispyrrole (+16, +32,

+48, +64).

92

Scheme 3.8 Possible fragmentation of an iso[4]LGE2-(N-acetyl-gly-lys-OMe)

aminal cross-link at m/z 853.5 (M + H+).

96

Scheme 3.9 Possible fragmentation of iso[4]LGE2-FluoTag aminal cross-link

with N-acetyl-gly-lys-OMe at m/z 1120.6 (M + H+).

100

Chapter 4

viii

Scheme 4.1 LG/isoLG modification of proteins 121

Scheme 4.2 Synthesis of iso[4]LGE2-pyrrole. 124

Scheme 4.3 Reaction of pure iso[4]LGE2-pyrrole with N-acetylcysteine. 130

Scheme 4.4 Possible electron transfer mechanism for pure iso[4]LGE2-pyrrole

reaction with N-acetylcysteine.

134

Scheme 4.5: Reduction of TEMPO by ferrous iron. 140

Scheme 4.6: Possible electron transfer mechanism for pyrrole oxidized reaction 143

Scheme 4.7 Initiate reactions of 3-ethyl-2,4-dimethylpyrrole 144

Scheme 4.8 Preparation of 3-ethyl-1,2,4-trimethylpyrrole (MP) 145

Scheme 4.9 Initiate reactions of 3-ethyl-1,2,4-trimethylpyrrole with TEMPO

and TMAO

145

ix

LIST OF TABLES

Chapter 2

Table 2.1 Molecular weight of iso[4]LGE2-modified and unmodified

calpain-1 peptide “SVTGAKQVNY”.

42

Table 2.2 Tryptic peptides from GAPDH modified by fluorescently labeled

iso[4]LGE2

51

Chapter 3

Table 3.1 Optimized parameters for MALDI-TOF mass spectrometer. 111

Table 3.2 Optimized parameters for triple quadrupole mass spectrometer. 112

Chapter 5

Table 5.1 Sequential adaptation of hybridoma cells to serum free medium 178

x

LIST OF FIGURES

Chapter 1

Figure 1.1 Pathological effects of LGs and isoLGs. 6

Figure 1.2 Overview of monoclonal antibody production, see text for detailed

description (Copyright from wikipedia commons).

21

Chapter 2

Figure 2.1 Treatment of calpain-1 with 10 equivalents of iso[4]LGE2 for

various times.

38

Figure 2.2 Treatment of calpain-1 with various equivalents of iso[4]LGE2 for

30 min

39

Figure 2.3 MS of iso[4]LGE2-modified and unmodified calpain-1 peptide

“SVTGAKQVNY” with the modification site at K280.

42

Figure 2.4 Tandem MS characterization of the m/z 690.88 doubly charged

ion from a MS scan of chymotrypsin digested iso[4]LGE2-

modified calpain-1 showed a series of fragment ions that

unambiguously identify an iso[4]LGE2 modification on the lysyl

residue (K280).

43

Figure 2.5 Ball-and-stick and cartoon 3D model structures for the catalytic

subunit of calpain-1(pdb: 2ary).

45

Figure 2.6 The active sites of human minicalpain 1 are shown in stick

representation and labeled (left panel).

46

Figure 2.7 Western analysis of iso[4]LGE2 (50 equivalents) modified calpain- 47

xi

1 after reaction for 1h, 3h, 12h and 24h. Left panel: developed

with anti-calpain-1 catalytic subunit primary antibody.

Figure 2.8 A ball-and-stick and cartoon 3D model structure for catalytic

subunit of GAPDH (pdb: 1j0x).

52

Figure 2.9 Coomassie blue staining of 10 equivalents fluorescently labeled

iso[4]LGE2 modified GAPDH after incubation for various times

prior to addition of excess glycine.

53

Chapter 3

Figure 3.1 MALDI-TOF analysis of the reaction mixture from N-

acetylglycyllysine methyl ester and iso[4]LGE2 (molar ratio =

200:1) at 25°C for 4 days.

90

Figure 3.2 LC-ESI for the product mixture from the reaction of N-

acetylglycyllysine methyl ester with iso[4]LGE2 (molar ratio =

200:1 at 25 °C for 4 days) with selected ion recording for aminal

cross-link, lactam-H2O and bispyrrole. A) TIC, B) aminal cross-

link m/z 854.1 (+ H+), C) lactam-H2O m/z 574.7 (+ H+), D)

bispyrrole m/z 1150.5 (+ H+).

91

Figure 3.3 MALDI-TOF spectra: panel A and B for 36 min HPLC fractions,

panel C for 30 min HPLC fraction, panel D for 41 min HPLC

fraction of products from the reaction of N-acetylglycyllysine

methyl ester with iso[4]LGE2 (molar ratio = 200:1 at 25 °C for 4

and 15 days).

93

xii

Figure 3.4 Top: Structures of A2E (1), the bisoxirane (2) and nonaoxirane

(3). Bottom: ESI mass spectrum of A2E after illumination with

blue light (430 nm; 10 min) to yield a mixture of various

oxygenated derivatives resulting in a series of ions that differ in

m/z by 16 owing to the addition of oxygen atoms to A2E. The

addition of oxygen presumably to form epoxides, occurred at

carbon-carbon double bonds of A2E, measured by Micromass

ESI-Q-TOF.

94

Figure 3.5 ESI-MSMS of molecular ions at A) m/z 835 (+ H+), and B) m/z

853 (+ H+) corresponding to an iso[4]LGE2-(N-acetyl-gly-lys-

OMe) aminal cross-link and its dehydrated form.

96

Figure 3.6 MALDI-TOF analysis of the product mixture from the reaction of

N-acetylglysyllysine methyl ester with iso[4]LGE2-FluoTag

(molar ratio = 200:1) at 25 °C for 15 days.

98

Figure 3.7 ESI-MSMS of molecular ions at A) m/z 1102.5 and B) m/z 1120.6

corresponding to a iso[4]LGE2-FluoTag-(N-acetyl-gly-lys-OMe)

aminal cross-link adduct and its dehydrated form.

100

Figure 3.8 MALDI-TOF analysis of the product mixture from the reaction of

β-amyloid(11-17) peptide “EVHHQKL” with iso[4]LGE2 (molar

ratio = 200:1) 25 °C for 7 days.

101

Figure 3.9 LC-ESI for the reaction mixture from β-amyloid(11-17) peptide

and iso[4]LGE2 (molar ratio = 200:1 at 25 °C for 7 days) with

selected ion recording for bispyrrole and aminal cross-links. A)

103

xiii

TIC, B) bispyrrole m/z 804.6 (+3), m/z 603.7 (+4), C) aminal

cross-link m/z 1058.2 (+2).

Figure 3.10 MALDI-TOF spectra: panel A and B for 32 min HPLC fractions,

panel C for 22 min HPLC fraction of products from reaction of the

β-amyloid(11-17) peptide “EVHHQKL” with iso[4]LGE2 reaction

(molar ratio = 200:1 at 25 °C for 7 and 15 days).

105

Figure 3.11 MALDI-TOF analysis of the product mixture from the reaction β-

amyloid (EVHHQKL) peptide with iso[4]LGE2-FluoTag (molar

ratio = 200:1) at 25 °C for 15 days.

107

Chapter 4

Figure 4.1 MALDI-TOF analysis of iso[4]LGE2-pyrrole and oxidation

products generated upon short term exposure of the reaction

mixture from iso[4]LGE2 with N-acetylglycyllysine methyl ester

in the presence of dithionite to air or longer term aeration with

oxygen before or after removal of dithionite.

125

Figure 4.2 LC-ESI for the purification of iso[4]LGE2-pyrrole with selected

ion recording for iso[4]LGE2-pyrrole.

126

Figure 4.3 Time course of the oxidative reactions of pure iso[4]LGE2-pyrrole

under air at 25 °C for 8 days.

128

Figure 4.4 MALDI-TOF of pure iso[4]LGE2-pyrrole and its oxidation

products: lower trace = pure iso[4]LGE2 -pyrrole, upper trace =

after incubation under air at 25 °C for 8 days.

129

xiv

Figure 4.5 MALDI-TOF spectra of pure iso[4]LGE2-pyrrole reaction with N-

acetylcysteine under air.

131

Figure 4.6 MALDI-TOF of pure iso[4]LGE2-pyrrole and its reaction with N-

acetylcysteine.

132

Figure 4.7 MALDI-TOF MS of pure iso[4]LGE2-pyrrole reaction with N-

acetylcysteine under air at 25 °C for 8 days.

133

Figure 4.8 MALDI-TOF MS of pure iso[4]LGE2-pyrrole and its oxidation

products: lower trace = pure iso[4]LGE2-pyrrole, upper trace =

after exposure to 2 mol% APS and 1 mol% TEMED at 37 °C for

2 h.

139

Figure 4.9 MALDI-TOF MS of pure iso[4]LGE2-pyrrole and its oxidation

products: lower trace = pure iso[4]LGE2-pyrrole, upper trace =

after exposure to 10 mol% TEMPO at 37 °C for 3 h.

141

Chapter 5

Figure 5.1 Mouse serum immune responses (1:10,000 dilutions) comparison

with pre-immune serum.

162

Figure 5.2 SDS-PAGE analysis of purified anti-iso[4]LGE2 mAb with protein

G column.

165

Figure 5.3 Binding inhibition of protein G purified anti-iso[4]LGE2 mAb

from serum free medium by iso[4]LGE2-HSA, iso[4]LGE2-KLH,

LGE2-BSA, LGE2-KLH, CEP-BSA, CEP-HSA, BSA and HSA.

166

Figure 5.4 Comparative Western analyses using anti-iso[4]LGE2 mAb against 167

xv

iso[4]LGE2-BSA, a standard protein containing the iso[4]LGE2

modification.

xvi

ACKNOWLEDGEMENTS

I wish to express my deepest sense of gratitude and respect to my research advisor Dr.

Robert G. Salomon for his expert guidance, constant encouragement, and the amount of

time he spent to help me to become a professional researcher over the years. He provided

me with the best possible facilities, collaborations, opportunities and challenging tasks,

which enable me to enhance my research aptitude and build a concrete foundation for my

future goals.

I also wish to express my deepest thanks to Dr. John W. Crabb, Cole Eye Institute,

Cleveland Clinic Foundation, who co-supervised me in last three years of my graduate

education. His invaluable guidance in protein chemistry and mass spectrometry, and

active participation in the research projects enabled me to achieve many of the results in

this thesis. Thanks to people in his research group, Lei Zhang and Jack Crabb for

valuable training and many insightful discussions. Special thanks to Dr. Geeng-Fu Jang,

he was there throughout my studying and always willing to help whenever I had

questions or problems. He has often helped me come out of one or more of the tight

corners in my projects. Above all I thank them for their friendship and guidance.

I would like to thank my committee members, Dr. Michael G. Zagorski, Dr. James

Burgess, Dr. Rajesh Viswanathan, Dr. John Crabb for their valuable time, efforts and

suggestions in my candidate qualification and thesis.

I would like to thank Dr. Salomon’s group, former and current members: Dr. James

Laird, Dr. Mikhail D. Linetsky, Dr. Xiaodong Gu, Dr. Jaewoo Choi, Dr. Li Hong, Dr.

Yunfeng Xu, Dr. Hua Wang, Dr. Xiaoxia Z. West, Dr. Wenyuan Yu, Dr. Yu Zhang, Dr.

xvii

Yalun Cui, and Junhong Guo, Liang Xin, Yuanyuan Qian, Guangyin Wang, Nicholas D.

Tomko, Yu-Shiuan Cheng for their thoughtful discussions and their friendship.

I would like to thank Dr. Irina Pikuleva of Department of Ophthalmology and Visual

Sciences for giving me the opportunity to work in her lab, and Dr. Casey Charvet for

valuable training of protein modification at the beginning of my research. I also wish to

thank Denice Major of Hybridoma Cole Facility of Visual Sciences Research Center

(VSRC) on the project of monoclonal antibody development.

I would like to thank Dr. Jim Faulk and other staff members in Chemistry Department

in Case Western Reserve University for their help in numerous occasions.

Finally, this thesis is dedicated to my family, especially my mom, my husband, for

their constant support, endless love and encouragement throughout the course of my

education.

xviii

LIST OF ABBREVIATIONS AND ACRONYMS

Abbreviations and Acronyms Equivalent

AA arachidonic acid AAA amino acid analysis AA-PC 2-arachidonyl-phosphatidylcholine Aβ amyloid β ABTS 2,2-azino-bis (3-ethylbenzthiazoline-6-

sulphonic acid diammonium salt AcOH acetic acid AMD age-related macular degeneration APS ammonium persulfate BSA bovine serum albumin CDCl3 deuterated chloroform CEP carboxy ethylpyrrole 13C-NMR carbon magnetic resonance CDI 1,1'-Carbonyldiimidazole CID collision-induced dissociation COX cyclooxygenase Cys cysteine DBD-ED 4-(N,N-Dimethylaminosulfonyl)-7-(2-

aminoethylamino)-2,1,3-benzoxadiazole kDa or kD kilodalton DCC dicyclohexylcarbodiimide

DMEM dulbecco’s modified eagle’s medium

xix

DMSO dimethyl sulfoxide DNA deoxyribonucleic acid DPDS 2,2'-Dipyridyldisulfide DTT dithiothreitol EDCI N-(3-Dimethylaminopropyl)-N′-

ethylcarbodiimide hydrochloride EDTA ethylenediaminetetraacetic acid EGTA ethylene glycol tetraacetic acid ELISA enyzeme-linked immunosorbant assay ESI electrospray ionization ETN ethanolamine FBS fetal bovine serum FTICR fourier transform ion cyclotron resonance GAPDH glyceraldehyde-3-phosphate dehydrogenase Gly glycine GSH reduced L-glutathione HAT hypoxanthine-aminopterin-thymidine 1H-NMR Proton magnetic resonance HNE 4-hydroxy-2-nonenal HoBt 1-hydroxybenzotriazole HPLC high performance liquid chromatography HRP horseraddish peroxidase HSA human serum albumin HUVEC human umbilical vein endothelial cell

xx

Hz hertz IC50 inhibitor concentration at the 50% absorbance

value IgG immunoglobin G iso[4]LGE2 iso[4]levuglandin E2 isoLG isolevuglandin isoLGE2 isolevuglandin E2

isoLGD2 isolevuglandin D2 KLH keyhole limpet hemocyanine

LC liquid chromatography LC/MS-MS liquid chromatography-coupled electrospray

tandem mass spectrometry LDL low-density lipoprotein LG levuglandin LGD2 levuglandin D2 LGE2 levuglandin E2 Lys lysine mAb monoclonal antibody

MALDI-TOF matrix assisted laser desorption ionization-

time of flight

NAPE-PLD N-acyl phosphatidylethanolamine-hydrolyzing phospholipase D

MeOH methanol MRM multiple reaction monitoring MS/MS tandem mass spectroscopy

xxi

NEM N-ethyl morpholine

NMR nuclear magnetic resonance OHdiA 4-oxo-heptanedioic amide ox oxidized pAb polyclonal antibody PAGE polyacrylamide gel electrophoresis PBS phosphate buffered saline PE phosphatidylethanolamine PEG polyethylene glycol PGs prostaglandins PGD2 prostaglandin D2 PGE2 prostaglandin E2 PGG2 prostaglandin endoperoxide G2 PGH2 prostaglandin endoperoxide H2 PM pyridoxamine ppm parts per million PUFAs polyunsaturated fatty acid(s) PVDF polyvinylidene fluoride QTof2 quadrupole-time of flight Rf retention factor RBC red blood cell ROS reactive oxygen species RPE retinal pigmented epithelium SET single electron transfer

xxii

SDS sodium dodecyl sulfate TEMED tetramethylethylenediamine TEMPO tetramethylpyrrolidine-N-oxide TFA trifluoroacetic acid TLC thin layer chromatography

TM trabecular meshwork TMAO trimethylamine-N-oxide TMS trimethyl silyl UV ultraviolet

xxiii

Part I: Isolevuglandin-protein Cross-linking: Structure and Mechanism

Part II: Generation and Characterization of a Monoclonal

Isolevuglandin[4]E2-protein Adduct Antibody

Abstract

By

WENZHAO BI

IsoLG modification of proteins is associated with loss-of-function, cross-linking and

aggregation. The sites of 1:1 adduct formation and the types and extent of cross-linking

between subunits of the multi subunit proteins calpain-1 and glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) by iso[4]LGE2 were characterized mass spectroscopically and

by SDS-PAGE. Modification of one or two lysyl residues calpain-1 suffices to abolish

activity. LC-MS/MS analysis established that Lys280 is a target of isoLG adduction to

calpain-1, Lys3 and Lys249 are targets of fluorescently labeled iso[4]LGE2 adduction to

GAPDH. Hetero- and homo-oligomers of calpain-1 catalytic and regulatory subunits

were detected by SDS PAGE.

N-Acetyl-glycyl-lysine methyl ester and β-amyloid(11-17) peptide “EVHHQKL”

were used as models for characterizing the cross-linking of protein lysyl residues

resulting from adduction of iso[4]LGE2. Aminal, bispyrrole and trispyrrole cross-links of

two model peptides, were identified and fully characterized by mass spectrometry, and a

large variety of derivatives of the bispyrrole cross-link containing one or more additional

atoms of oxygen were discovered. In sharp contrast with the dipeptide, the reaction of

xxiv

“EVHHQKL” with iso[4]LGE2 strongly favored bispyrrole versus aminal crosslink

formation. It is tempting to speculate that the “EVHHQKL” peptide-pyrrole modification

forms aggregates that favor the dimerization to produce bispyrrole since β-amyloid tends

to spontaneously oligomerize.

I discovered that the pure LG/isoLG-pyrrole derivative of the N-acetyl-lys-gly methyl

ester dipeptide could be produced in the presence of dithionite, an oxygen scavenger. The

oxidation and dimerization of the pure pyrrole is promoted by oxygen but exhibits an

induction period that can be eliminated by various catalysts, e.g., the stable free radical

tetramethylpyrrolidine-N-oxide or by trimethylamine-N-oxide.

In the presence of N-acetylcysteine, incubation of iso[4]LGE2-pyrrole at neutral pH

under air showed no pyrrole-to-pyrrole cross-linking. Instead a series of new ions was

produced corresponding to pyrrole cysteine adducts and a series derivatives incorporating

one or more atoms of oxygen. It is suggested that the thiol inhibits the pyrrole

dimerization by intercepting pyrrole cation radical generated by electron transfer to

oxygen.

An anti-iso[4]LGE2 mAb was prepared that exhibits high structural specificity for

detecting iso[4]LGE2–protein adduct epitope, and no significant cross-reactivity with

analogs including carboxyethylpyrrole (CEP) or LGE2-protein adduct epitopes.

xxv

CHAPTER 1

INTRODUCTION

1.1 Lipid Peroxidation

Lipids are essential and major components of cell membranes, and play important

roles in signaling pathways,1-2 energy storage, and gene expression.3-4 Oxidative

degradation of lipids occurs when there is an increase in the level of reactive oxygen

species which damages the cell membrane and leads to subsequent cell death. The

oxidation of polyunsaturated fatty acids (PUFAs) by enzymatic or non-enzymatic

reactions with molecular oxygen is termed “lipid peroxidation”.

Lipid peroxidation products have been implicated in a number of disease

conditions such as amyotrophic lateral sclerosis,5 age-related macular degeneration

(AMD),6-7 end-stage renal disease (RD),8-9 Alexander’s,10 Alzheimer’s,11-13 and

Parkinson’s14 diseases. It is also suspected to involve in the pathogenesis of

atherosclerosis (AS),15-16 antiphospholipid antibody syndrome,17 rheumatoid

arthritis,18 inflammatory bowel disease19 and multiple sclerosis.20 However, our

understanding of these processes on a molecular level remains primitive. Research in

Dr. Salomon’s group is making major strides to fill this gap. Our approach aims to

understand the biologically important chemistry of oxidized lipids and to determine

the extent and consequences of this chemistry in the pathogenesis of human diseases.

1.2 Enzymatic Pathways

Arachidonyl phospholipids, e.g., phosphatidyl choline with a palmityl or steryl

ester on the C1 hydroxyl and a arachidonyl ester on the C2 hydroxyl (AA-PC), a

linear twenty carbon methylene interrupted polyunsaturated fatty acid (C20:4ω6)

ester, is one of the most abundant polyunsaturated phospholipids in human low

2

density lipoprotein (LDL). It undergoes lipid peroxidation by two different pathways,

enzymatic (cyclooxygenase or lipoxygenase) and free radical mediated processes,

generating a vast array of biologically active oxidized products. The free fatty acid is

the substrate for enzyme-mediated peroxidation, while the free radical pathway

operates predominantly on esters.21

(CH2)3COOH

C5H11

710

13

AA

2O2 cyclooxygenase

(CH2)3COOHC5H11

OH

8

PGH2

OHC

(CH2)3COOHC5H11

OH

8

OO

(CH2)3COOHC5H11

OH

8

O

HO

1512

1

PGE2

O

LGE2

1

129

(CH2)3COOHC5H11

OH

8OHC

LGD2

1

129

O

(CH2)3COOHC5H11

OH

8HO

O

1512

1

PGD2

Scheme 1.1 Cyclooxygenase oxidation of AA generates PGs and LGs via

rearrangement of PGH2.

In the cyclooxygenase pathway, arachidonic acid (AA) is oxidized in a

stereospecific manner leading to the formation of the prostaglandin endoperoxide

3

PGH2. Besides enzyme-mediated transformations, a nonenzymatic spontaneous

rearrangement of PGH2 generates prostaglandins PGD2 and PGE2 (scheme 1.1).

Dr. Salomon made the discovery22-24 that PGH2 undergoes rearrangement via an

alternative pathway to form two γ-ketoaldehydes or levuglandins named LGE2 and

LGD2 (Scheme 1.1). The word levuglandin comes from the combination of

levulinaldehyde and prostaglandin because they are derivatives of levulinaldehyde

with prostanoid side chains.25 This rearrangement involves the intramolecular

migration of a bridgehead hydride to an electron deficient incipient methyl group with

concomitant cleavage of a C-C and O-O bond as shown in Scheme 1.2.26-27

Scheme 1.2 Schematic representation of rearrangement of PGH2 to levuglandin.

1.3 The Free Radical Pathway

Unlike the enzymatic pathway that converts free AA to an endoperoxide

intermediate, the free radical pathway mainly oxidizes AA-PC directly, rather than

free AA.25 It produces stereo and structural isomers of prostaglandins28 which were

named isoprostanes.29 Consequently, the free radical pathway is also called the

isoprostane pathway. This pathway also generates racemic mixtures of 16 LG

diastereomers, which a referred to collectively as isoLGE2 and isoLGD2 (Scheme 1.3).

Free radical-induced cyclooxygenation also generates 8 diastereomers of each of 6

4

structurally isomeric analogues that we designate collectively as iso[n]LGs. They are

structurally unique and give protein adducts that can only be products of free radical-

induced lipid oxidation.

(CH2)3COOR

C5H11

OH

OHC

(CH2)3COOR

C5H11

OH

O

OO

O

OO

OC

(CH2)nCH3

OP

O N(CH3)3O O

C5H11

(CH2)3COOR

OH

OO

OO

OO

OH

(CH2)3COOR (CH2)3COOR

OH

C5H11

(CH2)3COOR

OHOHC OHC OHC

OH


OH

C5H11

O OO

C5H11

C5H11

(CH2)3COOH

C5H11

OH

N

H3C

(CH2)3COOH

OH

OH

(CH2)3COOH(CH2)3COOH

OH

C5H11

C5H11

NNN

H3CH3C

H3C

NH2 NH2NH2 NH2

AA-PC

8-epi-PGH2-PC

12

8-epi-LGE2-PC

iso[4]-PGH2-PC

LGE2-pyrrole

-2 H2O protein -2 H2O protein-2 H2O protein -2 H2O protein

proteinprotein

proteinprotein

8

710

13

8

iso[10]-PGH2-PC iso[7]-PGH2-PC

iso[4]-LGE2-PC iso[10]-LGE2-PC iso[7]-LGE2-PC

iso[4]-LGE2-pyrrole iso[10]-LGE2-pyrrole iso[7]-LGE2-pyrrole

2 O22 O2 2 O2

2 O2

OHC (CH2)3COOR

C5H11

OH

(CH2)3COOR

OH

OHCOHC OHC

OH


OH

C5H11

C5H11

(CH2)3COOH

C5H11

OH

N(CH2)3COOH

OH

OH

(CH2)3COOH(CH2)3COOH

OH

C5H11

C5H11

NNN

NH2NH2

NH2 NH2

O OO O

H3C H3CH3C

H3C

12

8-epi-LGD2-PC

LGD2-pyrrole

-2 H2O protein-2 H2O protein

-2 H2O protein -2 H2O protein

proteinprotein

proteinprotein

8

iso[4]-LGD2-PC iso[10]-LGD2-PC iso[7]-LGD2-PC

iso[4]-LGD2-pyrrole iso[10]-LGD2-pyrrole iso[7]-LGD2-pyrrole

Scheme 1.3 Generation of isolevuglandins and isolevuglandin-protein adducts during

free radical oxidation of AA-esters

The studies described in this thesis focused on iso[4]LGE2, an isomer formed

exclusively through the free radical oxidation of AA-PC. LGs and isoLGs are highly

reactive aldehydes and initially modify the ε-amino group of lysyl residues in proteins

within seconds30 to form Schiff base intermediates that subsequently afford pyrroles

5

by dehydration.31 The pyrroles are readily oxidized producing lactams and

hydroxylactams.32-34 LGs/isoLGs covalent binding with proteins is accompanied by

cross-linking35 and hence polymerization of proteins orders of magnitude more

rapidly than other reactive arachidonate metabolites such as malondialdehyde and 4-

hydroxynon-2-enal. LGs/isoLGs also cause DNA-protein cross-links (DPCs) which

are repair-resistant and were shown to be relevant to cell killing (Scheme 1.4).36

O

O

R2

DNA

R1

NH2

Levuglandin

ON

R2Schiff base

Protein

R1

Protein NH2

N

H3C OH

NH

Protein-protein Crosslink

Protein

Protein

R1

R2

ON

R2Protein

R1

NHProtein

H

N

H3C OH

NH

DNA-protein Crosslink

Protein

DNA

R1

R2

ON

R2Protein

R1

NHDNA

H

N

H3C

Pyrrole

Bispyrrole Crosslink

Protein

O2 ROO

N

H3C

Protein

OH

OHydroxylactam

+

N

H3C

Protein

OLactam

R1

R2

R1

R2

R1

R2

N

N

R1

R2

R2

R1

Protein

Protein

H2O

Scheme 1.4 Formation of LG-protein adducts, protein-protein and DNA-protein

cross-links.

6

LGs and isoLGs have since been implicated in a number of diseases such as age-

related macular degeneration (AMD), atherosclerosis (AS) and cardiovascular disease

(CVD).25, 37-39 The possible pathological effects of LGs and isoLGs are summarized in

Figure 1.1.

Arachidonyl Phospholipids

Prostaglandin Endoperoxide

PGH2

Endoperoxides from phosphlipids

LGs

isoLGs

PGD2

PGE2

Proteins Covalent Adducts

Protein-protein Cosslinks

DNA-protein Cosslinks

Malfunction of protein and DNA

Cell death and tissue damage

Aggregation & proteasomal malfunction

Figure 1.1 Pathological effects of LGs and isoLGs.

1.4 Biological Activities of Levuglandins and Isolevuglandins

1.4.1 The effects of LGs/isoLGs on protein activity

IsoLG-mediated loss of protein activity has been reported.40 IsoLG, generated in

the epicardial border zone of the canine healing infarct, potentiated inactivation of

cardiac Na+ channels in human embryonic kidney (HEK)-293 cells and cultured atrial

(HL-1) myocytes, suggesting Na+ channel dysfunction evoked by lipid peroxidation

was a candidate mechanism for ischemia-related conduction abnormalities and

arrhythmias.41 In addition to the sodium channel, addition of synthetic isoLG/LG to

an atrial tumor myocyte cell line, AT-1, led to a pronounced dose-dependent

inhibition of the inward rectifying potassium current induced by a -40 mV voltage

step (IC50 = 2.2 μm).42 Oxidative stress reduces the activity of isolated Na+, K+-

ATPase enzymes. In the pioneering study of Shattock et al.,43 research was performed

to determine the effects of oxidant stress on the Na(+)-K+ pump current with isolated

rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Through

7

photoactivation of rose bengal, singlet oxygen and superoxide were generated and

isolated cells were exposed to the induced oxidant stress. Results showed that the

Na+-K+ pump current was inhibited by oxidant stress at all voltages and this inhibition

may contribute to ischemia/reperfusion injury.43 The enzyme activity of purified

RNase A and glutathione reductase was found to decrease following a treatment with

isoLGE2 in vitro. An isoLG scavenger, pyridoxamine, protects enzymes against loss

of activity caused adduction by isoLGE2.44

The first direct evidence linking isoLG modification of a specific protein and loss

of enzyme activity in vivo involved the protease activity of calpain-1. Elevated levels

of the enzyme were detected in glaucomatous trabecular meshwork (TM)45 compared

to nonglaucomatous control (calpain-1 extracted from normal eyes), but calpain-1

activity in the glaucomatous trabecular meshwork was greatly reduced, about 50%

less activity than the same amount of protein from normal TM. Elevated levels of

iso[4]LGE2 protein adduct were also observed in glaucomatous astrocytes isolated

from human optic nerve.46

In an in vitro study, iso[4]LGE2 modified-calpain-1 was ubiquitinated by a HeLa

cell-derived system and the ubiquitinated calpain-1 could be efficiently recognized by

HeLa cell-derived constituted proteasome 26S. Dynamic light scattering studies that

were performed to monitor the size of aggregates confirmed the presence of large

aggregates that correspond to ubiquitinated iso[4]LGE2-modified calpain-1.

Furthermore, these aggregates were refractory toward degradation. Human eyes

possibly lack the capacity to degrade iso[4]LGE2-modified proteins, resulting in their

8

accumulation.45 Glaucoma is thought to be caused in part by an increase in intraocular

pressure (IOP), however normal IOP glaucoma is also common. Pressure increases

either when too much aqueous humor fluid is produced or by decreased aqueous

humor outflow. The trabecular meshwork is responsible for most of the outflow of

aqueous humor. The inhibition of proteasome activity using epoxomicin on cultured

human primary TM cells indicated increased calpain-1 accumulation, owing to

protein modification by isoLGs. This could contribute to glaucoma pathophysiology

by decreasing the ability of the TM to modulate outflow resistance.45 Given the

importance of ubiquitination for proteasomal clearance of LG/isoLGs modified

proteins, it is noteworthy that ubiquitin was shown to form adducts with LGE2.45

The mitochondrial enzyme cytochrome P450 27A1 (CYP27A1) plays a very

important role in the maintenance of cholesterol homeostasis, bile acid biosynthesis,

and activation of vitamin D347 by catalyzing the C27-hydroxylation of cholesterol and

other sterols. Recently CYP27A1 was found to be expressed in the retina48-49 and

shown to be the major contributor to enzymatic degradation of cholesterol.50

Treatment of CYP27A1 with iso[4]LGE2 in vitro diminished enzyme activity in a

time- and phospholipid-dependent manner.51 To model membrane insertion,

CYP27A1 was reconstituted into various PLs. It was found that the enzyme is

modified even when other biological amines are present and iso[4]LGE2

concentrations are relatively low (2-folder molar excess). Liposomes of various

composition were as follows: bovine retinal mitochondrial PLs, a DLPC (1,2-

dilauroyl-snglycero-3-phosphocholine) and DOPE (1,2-dioleoyl-snglycero-3-

9

phosphoethanolamine) mixture (4:3, w/w) that approximates the ratio of

phosphatidylcholine to phosphatidylethanolamine found in human hepatic

mitochondria, or DLPC that contains no free amines and is a standard model PL. In

this set of experiments, the ratio of iso[4]LGE2 was a 2-fold molar excess. Enzyme

activity was decreased sharply by 75% at 5 min and the P450 content decreased only

by 25%, indicating that modification impaired activity without completely denaturing

the protein. Most modification and loss of activity occurred within 15 min of

incubation of a 2-fold molar excess of iso[4]LGE2 with the protein. In contrast,

incorporation of the protein into liposomes containing ethanolamine phospholipids

protected against modification owing to interception of isoLGs by the primary amino

groups of the ethanolamine phospholipids.

Lys 358-isoLG adduct in CYP27A1 was found in human AMD retinal sample. To

study the effect of Lys 358 modification on the enzyme activity, mutations were

introduced to the wild-type (WT) CYP27A1. CYP27A1 Lys 358 was mutated to

either hydrophobic Leu to mimic removal of the positive charge or to Arg to conserve

the charge but abolish iso[4]LGE2 modification because Arg is at least 1,000-fold less

susceptible than Lys to modification by isoLGs. The catalytic activity of the Lys 358

CYP27A1 mutant was characterized before and after isoLG treatment. It was

confirmed that modification of Lys 358 by isoLG is the major contributor to isoLG-

associated loss of CYP27A1 activity. This was first study to conduct the measurement

of enzyme activity and simultaneously correlate the isoLG modification to specific

locations in the primary sequence. The Lys 358-isoLG adduct in CYP27A1 was found

10

in human retina from individuals afflicted with age-related macular degeneration,52

providing direct evidence that isoLG adduction impairs enzyme activity and

supporting the hypothesis that isoLG modification of CYP27A1, potentially impairs

cholesterol homeostasis in the retina.52

1.4.2 LGs/isoLGs effect on the interaction of histones with DNA

DNA-protein cross-linking by a levuglandin was first observed upon treatment of

V79 Chinese hamster lung fibroblasts or nuclei with LGE2.36 DNA in the cell lysate

was retained on a nitrocellulose filter, presumably owing to covalent DNA-protein

cross-linking. This was confirmed by proteolysis with proteinase K that allowed the

DNA to pass through the filter. DNA tightly coils around histones in cells. The lysine-

rich histones from calf thymus could form adducts with isoLG/LG. It was postulated

that this protein is a likely candidate for DNA-isoLG-protein cross-linking.53 Recently,

the formation of LG-lysine lactam adducts on histones were identified in RAW264.7

mouse macrophages, A549 cultured lung epithelial cells and rat liver, that is

dependent on COX-2 activity or COX-2 expression. With an internal LG-lysyl

standard, highest measurable amounts of adduct was detected on the H4 histone.

Adduction was blocked by a γ-ketoaldehyde scavenger (4-ethylsalicylamine), which

had no effect on COX-2 activity as measured by PGE2 production. Formation of LG-

histone adduct was associated with an increased histone solubility in aqueous NaCl

and after LG-histone adduct formation, histone-DNA binding was disrupted with an

increased DNA extraction in a salt solution. Chromatin access and transcription was

regulated by a “histone code comprised with complex patterns of lysyl acetylation and

11

methylation.54 It is reasonable to conclude that irreversible adduction of lysyl residues

could disrupt this code, or directly alter the access of DNA-interacting proteins.

Changes in histone modifications are known to result in altered DNA methylation,

deregulation of oncogenes, genomic instability, impaired DNA repair, and defects in

cell cycle checkpoints.55-56 Changes in lysyl modifications of H4 in particular are a

common hallmark of human cancers, and are associated with a global loss of DNA

methylation.57 Further elucidation of the effects of LG-histone adduction on histone

modification, DNA-histone interactions, and transcription are expected to increase the

understanding of the molecular mechanisms whereby COX-2 contributes to cancer

development and progression.

1.4.3 LGs/isoLGs covalently modify PE

Besides the ε-amino groups of protein lysyl residues, iso[4]LGE2 covalently binds

ethanolamine phospholipids in vitro to form covalent pyrrole adducts that were

oxidized in air to deliver stable lactam and hydroxylactam (HL) adducts.58 In lipid

extracts from human plasma and mouse liver, isoLG-lysoPE-HL derivatives of

isoLGs was detected through phospholipase A2 (PLA2)-catalyzed hydrolysis of

isoLG-diacyl-PE precursors.58 The formation of isoLG/LG-PE is of interest since

oxidatively modified phospholipids have been implicated in certain autoimmune

diseases. Treatment both of human embryonic kidney (HEK293) and human

umbilical vein endothelial cells (HUVEC) with isoLGE2 (15-E2-isoketal, isoK)

generated more modified PE (isoLGE2-PE) than modified protein. As internal

standard, isoLGE2-[2H4] ethanolamine was incubated with HEK293 cells and

12

isoLGE2-PE was quantified by LC/MS/MS after hydrolysis to isoLGE2-ethanolamine

by Streptomyces chromofuscus phospholipase D. Analysis of isoLGE2-protein adducts

as isoLGE2-lysyl-lactam adduct after protease digestion was performed on the protein

pellet using isoLGE2-[13C615N2]lysyl-lactam as the internal standard. Human

umbilical vein endothelial cells (HUVEC) were treated the same way. IsoLGE2-PE

induced cytotoxicity towards human umbilical vein endothelial cells (HUVEC) with

LC50 as 2.2 µM. These observations indicate that cellular PE is a significant target of

isoLGs.59

Another study showed that PE modified by isoLGE2 (isoLGE2-PE) induced THP-

1 monocyte adhesion to human umbilical cord endothelial cells. IsoLGE2-PE also

induced expression of adhesion molecules and increased MCP-1 and IL-8 mRNA in

human umbilical cord endothelial cells and this pyrrole-PE markedly altered

membrane curvature.60 To test if cells could degrade isolevuglandin modified

phosphatidylethanolamine (isoLG-PE), the stability of isoLG-PE in human embryonic

kidney 293 (HEK293) cells and in human umbilical cord endothelial cells (HUVEC)

was measured over time. HEK293 cells were incubated with isoLGE2 to generate

isoLG-PE and excess isoLG was removed by washing with DMEM. With isoLGE2-

[2H4] ethanolamine as internal standard, isoLGE2-PE levels were measured by LC-MS

after conversion to its phosphoglycero-ethanolamine derivative by base hydrolysis.

The stability of isoLGE2-PE in endothelial cells was assessed the same way. Cellular

isoLGE2-PE levels in HEK293 cells decreased more than 75% after 6 h. While in

13

HUVEC cells, the isoLGE2-PE levels rapidly dropped with less than 5% of isoLGE2-

PE present after 24 h.63

N-Acyl phosphatidylethanolamine-hydrolyzing phospholipase D (NAPE-PLD) is

a key enzyme for the hydrolysis of N-acyl phosphatidylethanoamine (NAPE)

including both NAPE and isoLGE2-PE with large aliphatic headgroups. It hydrolyzes

isoLGE2-PE to isoLGE2-ethanolamine. These results demonstrate that NAPE-PLD

contributes to the degradation of isoLGE2-PE and suggest that a major physiological

role of NAPE-PLD may be to degrade aldehyde-modified PE, thereby preventing the

accumulation of these harmful compounds.61

1.4.4 LG/isoLG scavengers

Pyridoxamine (PM) is a B6 vitamin which reacts with isoLGs 2000-fold faster

than the ε-amino groups of protein lysyl residues, making it an efficient scavenger of

this type of lipid peroxidation products.62 Therefore, PM and its analogues can be

used as isoLG/LG scavengers to protect proteins from modification and inactivation

by isoLGs/LGs and to determine the impact on pathophysiology of specifically

reducing the levels of isoLG/LG protein adduction. Treatment with PM and its

analogs, salicylamine and 5’-O-pentylpyridoxamine, had been shown to prevent

H2O2-mediated cytotoxicity in HepG2 cell culture and decrease isoLG-protein adduct

formation of ovalbumin exposed to isoLG. PM also prevented inhibition of RNase A

and glutathione reductase activity by the γ-ketoaldehyde isoLGE2. RNase activity and

glutathione reductase (GR) activity were determined by measuring the formation of

14

acid-soluble oligonucleotide63 and the initial rate of NADPH consumption,64

respectively. 44, 65

Recent research showed that exposure of mice to bright light leads to isoLG-

adduct formation in the retina.66 Pre-treatment of mice with PM greatly decreased

retinal isoLG adduct levels resulting from light exposure, and morphological changes

in photoreceptor mitochondria were not as pronounced as in untreated animals. This

study demonstrated a novel concept in vision research, i.e., that preventing damage to

biomolecules by lipid peroxidation products is a viable strategy to combat oxidative

stress in the retina. This novel therapeutic activity of PM may be applicable to

humans as PM has good drug-like properties and an excellent safety profile.66

1.4.5 Detection of biological adducts of LGs/isoLGs

Detecting free LG/isoLG adducts in vivo is complicated by their proclivity to

behave like superglue, adhering to normal physiological proteins within seconds. On

the other hand, there are two complimentary methods for quantifying LG/isoLG

adducts in vitro/vivo. One method utilizes antibody based approaches such as ELISA,

Western blotting, or immunohistochemistry. The other uses mass spectroscopic

detection of (1) LG/isoLG modified lysine12 or peptides51 derived from LG/isoLG

modified proteins or of (2) LG/isoLG modified ethanolamine phospholipids.58 The

first detection of LG/isoLG adducted proteins in vivo was in patients with

atherosclerosis or end-stage renal disease using ELISA measurements.39

Immunoassays with antibodies raised against protein adducts of LGs generated by

unambiguous chemical synthesis provided evidence for their presence in human and

15

mouse blood and tissues and enabled the discovery that free radical-induced oxidation

of arachidonyl phospholipids in low-density lipoprotein generates LGE225 as well as

stereo and structural isomers referred to collectively as isolevuglandins.38 A single-

chain antibody from a phage displayed recombinant ScFv library that bound a model

peptide adducted with synthetic isoLGE2 was isolated and characterized. Recognition

of isoLGE2-protein adducts by this anti-isoLGE2 adduct single-chain antibody is

essentially independent of the amino acid sequence of the adducted peptides or

proteins, and no cross-reactivity was detected with 4-hydroxynonenal or 4-

oxononanal adducts or with 15-F2t-isoprostane (8-iso-prostaglandin F2α).67 However,

cross reactivity with structurally isomeric isoLGs was not determined.

The second method for detection of isoLGE2-modified proteins utilizes the

sensitivity and specificity of liquid chromatography-mass spectrometry quantitation of

the excised LG-modified lysine following exhaustive proteolysis of LG/isoLG-

adducts in tissues. A heavy isotope labeled internal standard is added to the sample

for quantification.68 MS analysis revealed that the majority of LG/isoLG-protein

modifications are lactams and hydroxylactams generated by oxidation of the initial

pyrrole modifications.32 LC–MS/MS analysis also detects isoLG-

phosphatidylethanolamine adducts in human blood and mouse liver.58 Site-specific

post-translational protein modifications can be detected and quantified using mass

spectrometry-based multiple reaction monitoring (MRM) assay.69 With this method,

the isoLG-modified tryptic peptide AVLKETLR in a mitochondrial protein, Cyp27A1,

16

extracted from human retina was detected and shown to be an adduct of the lysyl ε–

amine group.51

1.5 Monoclonal Antibody Development

1.5.1 Antigens and antibodies

Substances that are recognized by B-cell receptors are termed antigens.

Classically, an antigen is defined as any substance that elicits an immune response in

a susceptible animal and is capable of binding with the specific antibodies generated.

Antigens are usually of a high molecular weight and are commonly proteins or

polysaccharides, although nucleic acids, lipids and peptides have also been reported to

function as antigens.70 Antibodies are produced by plasma B-lymphocytes and

function as a part of an immune system in mammals in the battle against disease.

Antibodies have a common structure of four peptide chains, consisting of two

identical light chains, of about 25 KDa and two identical heavy chains of about 50

KDa. Each light chain is bound to the heavy chain by a disulfide bond and a

combination of hydrogen bonds, salt linkages and hydrophobic bonds. Similarly

noncovalent interactions and disulfide bridges link two identical heavy and light chain

combinations to each other to form a basic four chain immunoglobulin structure.71

Antibodies (Ab)s play an important role when foreign antigens (Ag)s, such as

pathogens and toxins, are introduced into an organism and start a complex immune

response. Antibodies label the foreign molecule or pathogen for destruction and

removal in a lock-and-key type of mode. The basic principle of any immunological

technique is that an antibody will combine with its specific antigen to give a unique

17

Ab/Ag complex. For an efficient Ag/Ab interaction to occur the epitope must be

exposed and available for binding, alterations in the conformation of epitopes through

tissue processing, fixation, reduction and pH changes may affect the binding. In

modern medical diagnostics and basic research, antibodies have become crucial

molecules since it is possible to develop specific antibodies against almost any

component, from small drug molecules to intact cells.

1.5.2 Polyclonal antibodies

Prior to 1975, polyclonal serum was the only antibody option available.

Polyclonal antibodies refer to antibodies present in the crude serum of an immunized

animal, such as a mouse, rabbit, goat or hen, capable of recognizing one or several

different immunogenic epitopes of the administered immunogen. These antibodies

may be of different subclasses, but isolation of a single subclass, usually IgG, is

readily achieved. Polyclonal antibodies are often used since they are easily purified

from the blood of the mammal by chromatographic techniques. Their utilization is

quick and inexpensive, requiring little skill or technical expertise. They are

particularly useful when amplification of a signal from a target protein with low

expression is required as they recognize multiple epitopes on one protein. However,

despite these advantages a pAb raised against an antigen can bind several different

epitopes on the target. This increases the likelihood that pAbs cross-react with

biomolecules containing similar epitopes. Anomalous results and variability between

batches often leads to inconsistencies in results. Furthermore, the supply of pAbs is

limited for research purposes as the mammal is eventually killed.

18

1.5.3 Monoclonal antibodies and hybridoma technology

A major milestone in immunological research was the development of

monoclonal antibodies by Köhler and Milstein in 1975.72 This Nobel Prize winning

work (1984, physiology and medicine) revolutionized antibody production and today

it forms the basis of many diagnostic applications, disease therapies and basic

research.70, 73 Production of monoclonal antibodies (mAbs), in contrast with pAbs, is

more time consuming and expensive from pAb production and requires high

technology and extensive technical skills. Monoclonal antibodies are only one

subclass, usually IgG, allowing for selection of an appropriate secondary antibody for

detection. Large quantities of specific antibodies can be produced and their specificity

ensures that only one epitope is recognized on an antigen. This is extremely useful

when studying subtle protein alterations, and the antibodies are less likely to cross

react with other proteins and generally produce less background cross reaction.

Hybridoma technology involves fusion of a mouse myeloma cell line with mouse

spleen cells from an immunized donor in the presence of polyethylene glycol (PEG).74

The resultant cells are grown in a selective medium in which only hybridoma cells

can proliferate.75 The resultant hybridoma cell line possesses the immortal growth

properties of the cancerous plasma cells and the antibody secreting properties of the

normal B cells, thus creating an indefinitely growing cell line that produces large

quantities of antibodies with identical specificity.

The fusion is random and fused hybridoma cells need to be selected and isolated

from unfused B-lymphocytes and myeloma cells. The cells are cultured in

19

hypoxanthine-aminopterin-thymidine (HAT) medium. Aminopterin (A) blocks the de

novo biosynthesis of purines and pyrimidines which are essential for DNA synthesis.

When this pathway is blocked, cells use the salvage pathway utilizing hypoxanthine

(H) and thymidine (T), and this requires the activity of the enzymes thymidine kinase

(TK) and hypoxanthine-guanine phosphoribosyl transferase (HGPRT). The myelomas

used for the fusion lack HGPRT, so that unfused myeloma cells and myeloma cells

fused to other myeloma cells cannot proliferate in HAT medium. The unfused

splenocytes do possess HGPRT but have a limited lifetime and will die within two

weeks. The hybridoma cells grow effectively in the HAT medium. Many different

hybridomas are created during the fusion and every cell type produces a specific

antibody towards a wide range of antigens and not only the antigen used to immunize

the mouse. To identify the hybridomas that are specific for the antigen, cells are

distributed in 96-well plates and hybridoma supernatant is used in an enzyme-linked

immunosorbent assay (ELISA) to detect the wells containing antibodies that bind with

the antigen (positive wells) for subsequent cloning by limiting dilution.76 The method

essentially consists of diluting the cells and growing them at very low densities, often

in the presence of feeder cells, which supply growth factors. After screening, positive

wells containing only one visible clone are selected for another round of cloning.

After at least three rounds of cloning, the cell line is per definition monoclonal.

Cloning continues until a stable hybridoma, i.e., that does not lose the mAb-producing

ability, is achieved. The stable cloned hybridomas can then be expanded for large-

scale mAb production. Hybridomas can be frozen for storage and in this way an

20

unlimited supply of mAb’s can be maintained (Figure 1.2). mAb may also be stored

frozen but may be come nonspecific inactive with long term storage.

Figure 1.2 Overview of monoclonal antibody production, see text for detailed

description (Copyright from wikipedia commons).

21

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24

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45. Govindarajan, B.; Laird, J.; Salornon, R. G.; Bhattacharya, S. K. (2008)

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46. Govindarajan, B.; Junk, A.; Algeciras, M.; Salomon, R. G.; Bhattacharya, S.

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49. Liao, W. L.; Heo, G. Y.; Dodder, N. G.; Reem, R. E.; Mast, N.; Huang, S.;

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32

CHAPTER 2

ADDUCTION AND CROSS-LINKING OF CALPAIN-1 AND

GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE

(GAPDH) BY ISO[4]LGE2

2.1 Background

2.1.1 Purpose

The first characterization of the site of in vivo isoLG post-translational modification

was of the monomeric mitochondrial protein Cyp27A1. To elucidate isoLG-mediated

protein-protein cross-linking, the reaction of iso[4]LGE2 with multi subunit proteins,

calpain-1 and GAPDH, was investigated. In this study, we characterized sites of

modification and detected cross-linking between subunits of the multi subunit proteins

calpain-1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

2.1.2 Isolevuglandin modification of proteins.

The eye and brain contain high levels of polyunsaturated fatty acids (PUFAs), of

which arachidonic acid is one of the most abundant.1 Arachidonyl phospholipids (PLs)

undergo cyclooxygenase catalyzed oxidation to produce γ-ketoaldehyde levuglandins and

free radical-induced oxidation in vivo to form levuglandins (LGs) and their stereo and

structural isomers referred to collectively as isolevuglandins (isoLGs). LG and isoLG-

protein adducts in human serum have been fully characterized.2 Some isoLG isomers

such as iso[4]levuglandin E2 (iso[4]LGE2) are not produced through the enzymatic

cyclooxygenase pathway, but rather are only generated through free radical-mediated

oxidation.3 On the other hand, a few diastereomers of two isoLG structural isomers, e.g.,

levuglandins E2 and D2, are not only generated through free radical-initiated lipid

oxidation, but are also formed through the cyclooxygenase pathway.4 IsoLGs are a highly

reactive family of γ-keto aldehydes5 that react with free primary amines such as the ε-

amino group of lysine residues in proteins. They form covalent adducts with orders of

magnitude greater avidity than most other lipid oxidation products such as 4-

34

hydroxynonenal.6-8 These adducts incorporate the primary amino group in a pyrrole ring

that is readily oxidized to lactam and hydroxylactam derivatives. In the human eye,

isoLG adducted calpain-1 accumulates in the trabecular meshwork, and isoLG adduction

abolishes the enzyme’s activity.9 LG/isoLG-protein adduction has biological

consequences. For example, preferential adduction of isoLG to a specific Lys in the

mitochondrial enzyme Cyp27A1 abolishes its ability to metabolize cholesterol leading to

cholesterol accumulation and retinal pathology.10-11 Lysyl-LG/isoLG-protein adduction

also results in intermolecular protein-protein cross-linking. Pyrrole-pyrrole cross-link and

aminal cross-link structures depicted in Scheme 2.1 have been postulated for isoLG-

protein modifications, but direct molecular level evidence for the cross-link structure(s) is

lacking.12

C5H11

COO-PC

O

O

OH

OHC C5H11

(CH2)3COO-PCO

OH

isoPGH2-PC

isoLGE2-PC

COO-PC

PAPCR1

R2

OHN

CH3

hemiaminal

R1

R2

N

H3C

O2

N

H3C X

Opyrrole

isoLGE2-lactam, X = HisoLGE2-hydroxylactam, X = OH

(CH2)3COOH

C5H11

OH

O2O

O

OHiso[4]PGH2-PC

COO-PC

C5H11

OH

COO-PC

C5H11OHC

O

iso[4]LGE2-PC

PLA2

N

H3C X

O

iso[4]LGE2-lactam, X = Hiso[4]LGE2-hydroxylactam, X = OH

OH

COOH

C5H11

protein NH2

protein

protein

protein

protein

R1

R2N

H3C

pyrrole-pyrrole crosslink

protein

R1

R2N

H3C

protein

O2

OH

protein NH2

R1

R2

HO

N

CH3

aminal crosslink

protein

protein

R1

R2

OHN

CH3

protein

NH

-H2O

protein NH

Scheme 2.1 Postulated structures of LG and iso[4]LG protein adducts and cross-links.

35

2.1.3 Calpain-1.

Calpains are ubiquitous calcium-dependent cysteine proteases that mainly exist in two

forms in all tissues, µ-calpain and m-calpain. They belong to a superfamily of 14 cysteine

proteases. Activation of calpains in vitro requires the presence of calcium at micromolar

quantities for µ-calpain and millimolar quantities for m-calpain at neutral pH.13-14 Both

forms comprise a large subunit of about 80 kDa and a small subunit of about 30 kDa.

Increased calpain activity has been discovered in several neurological disorders, such as

spinal cord injury, ischemic brain injuries, stroke, and calpain activity has been

implicated in several neurodegenerative disorders such as Alzheimer’s disease and

multiple sclerosis.14 IsoLG-protein adducts accumulate in trabecular meshwork (TM)

tissue in individuals with primary open angle glaucoma. Inactive calpain-1 accumulates

in the TM in conjunction with massive levels of isoLG-modified protein. Treatment with

isoLG inactivates calpain-1. There are elevated levels of the protease calpain-1 in

glaucomatous TM based on western analysis. However, calpain-1 activity in

glaucomatous TM is only about 50% of that in controls. This paradox is explicable by the

fact that modification by isoLGs renders calpain-1 inactive. In addition, isoLG-

modification of calpain-1 promotes its ubiquitination and the formation of aggregates that

resist proteasomal processing9, 159, 159, 159, 15,9, 15 and presumably contribute to the impeded

aqueous humor outflow through the TM that results in elevated intraocular pressure. It

appears that calpain-1 is expressed to promote proteolysis of damaged proteins. However,

this function is defeated by isoLG-modification that renders it inactive. In addition,

isoLG modification can also make proteins resistant to proteolysis, e.g., by the 20S

36

proteasome.16 Thus, isoLG adduction presumably contributes to accumulation and

inactivation of calpain in the TM owing to aggregation and/or resistance to proteolysis.

2.1.4 GAPDH

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays a key role in the

glycolytic pathway.17 GAPDH catalyzes the oxidative phosphorylation of

glyceraldehyde-3-phosphate (G3P) in the presence of NAD+ and inorganic phosphate.

GAPDH is well conserved during evolution, being a protein with a native molecular

weight in the range of 140-150 kDa. It is composed of four identical of 35-37 kDa

subunits.18 The ubiquity and evolutionary conservation of GAPDH indicate a highly

important physiological function.19 Besides its traditional role in glycolysis, newly

recognized activities of GAPDH include regulation of the cytoskeleton,20 membrane

fusion and transport,21 glutamate accumulation into presynaptic vesicles,22 and activation

of transcription in neurons.23 Elevated levels of nuclear GAPDH are observed in

postmortem samples from patients with Parkinson's disease, Alzheimer's disease,

Huntington's disease and glaucoma, among others.24 4-Hydroxy-2-nonenal (HNE), a

major lipid peroxidation-derived reactive aldehyde, exerts an inhibitory effect on

GAPDH due to the modification of the cysteine residue (Cys-149) at the catalytic site

where it forms an HNE-cysteine Michael addition type adduct.25 Elevated iso[4]LGE2

protein adduct immunoreactivity is also found in glaucomatous astrocytes derived from

the glaucomatous optic nerve head.26

37

2.2 Results and discussion

2.2.1 In vitro modification of calpain-1 with iso[4]LGE2 abolishes its protease

activity.

The activity of calpain-1 extracted from glaucomatous trabeculectomy tissue is about

50% less than the same amount of protein extracted from normal trabecular meshwork

tissue in spite of the fact that the levels of calpain-1 in glaucotomous TM are greatly

elevated compared with those in normal TM.9 Previously, treatment of purified calpain-1

with isoLG in vitro was shown to result in loss of catalytic activity but the time course of

activity loss was not explored.9 To further characterize the relationship between

modification of the protein by isoLG and decrease in activity, enzymatic activity was

monitored after treatment of the protein in vitro with iso[4]LGE2. Exposure of calpain-1

with iso[4]LGE2 in vitro diminishes enzyme activity in a time- (Figure 2.1) and ratio-

dependent (Figure 2.2) manner.

Figure 2.1 Treatment of calpain-1 with 10 equivalents of iso[4]LGE2 for various times.

Left: SDS-PAGE of proteins showed 10 min, 30 min and 90 min modification. 90’C

represented control within 90 min, while 90’M represented modification within 90 min,

38

same for 30 and 10 min. Right: calpain-1 activity assay with different modification time

treatment.

Notably, the inhibitory effect was not linearly related to isoLG-protein ratio. Two

equivalents of isoLG had little additional inhibitory effect over a 1:1 molar ratio and the

inhibition caused by a 10 equivalents of isoLG to protein was less than twice that of 2

equivalents. This behavior suggests that a low level of modification by one or two

molecules of isoLG has a profound effect on activity, and that additional modifications

only provide minor additional inhibitory effects. The time dependence of the effect of

exposure of the protein to 10 equivalents of iso[4]LGE2 was examined. Enzyme activity

decreased by 50% by 30 min and gradually decreased to 10% by 90 min while the

activity of the control, treated with buffer alone, was unchanged. Analysis by

electrophoresis showed that within 10 min after treatment, both the calpain-1 catalytic

and small subunits migrated at slightly higher mass than the untreated protein presumably

because they had been covalently modified.

Control2 x 5 x 10 x2025

37

50

75

100

150

250

Figure 2.2 Treatment of calpain-1 with various equivalents of iso[4]LGE2 for 30 min.

Control2 x 5 x 10 x2025

37

50

75

100

150

250

KDa

equivalent of iso[4]LGE2

39

Left: SDS-PAGE of calpain-1 after treatment with 2, 5 or 10 equivalents of iso[4]LGE2.

Right: calpain-1 activity after treatment with 2, 5 or 10 equivalents of iso[4]LGE2.

The ratio dependence of the effect of exposure of the protein to a 2, 5 10 equivalents

of iso[4]LGE2 was also examined. Enzyme activity decreased sharply by 50% within 30

min. Analysis by electrophoresis showed that 30 min after treatment, both the calpain-1

catalytic and small subunits migrated slightly less than the untreated protein presumably

because they had been covalently modified.

In the pioneering study of Govindarajan et al.,9 isoLG modified calpain-1 was

precipitated by cold acetone, and resuspended in PBS for enzyme activity test. Our study

aims to find specific sites of modification by isoLG and to test enzyme activity at the

same time, so calpain-1 modification was performed in sodium acetate solution. The

reaction was quenched by addition of glycine and the mixture used directly for enzyme

activity assay and MS analysis.

2.2.2 Calpain-1 Lys280 is modified upon exposure to isoLGs.

IsoLG modification of proteins, e.g., calpain-1, is associated with loss-of-function,

cross-linking and aggregation. To elucidate isoLG-mediated protein-protein cross-linking,

the reaction of iso[4]LGE2 with calpain-1 was examined in detail. A site of isoLG

modification of calpain-1 was identified by peptide mapping and sequencing. Using LC-

MS and Mascot analysis one lysine residue in calpain-1 was identified that was converted

by iso[4]LGE2 into lactam derivatives. A full MS comparison of the iso[4]LGE2 modified

and unmodified calpain-1 chymotryptic peptide “SVTGAKQVNY” shows that K280 is

the modification site (Figure 2.3).

40

Iso[4]LGE2-treated calpain-1 was proteolyzed in-gel with chymotrypsin followed by

FTICR MS/MS. Peak lists from the acquired MS/MS spectra were submitted to the

Mascot data base search engine. Evaluation criteria for identification of the modified

peptides were as follows: first, only hits with a significant peptide score as reported by

Mascot’s scoring algorithm27 were considered. Second, the tandem MS spectra were

manually inspected for the candidates to confirm the existence of y and b series ions

upstream and downstream of the modified lysine. Third, the presence of multiple adduct

dehydration and oxidation states was sought (Table 2.1S), e.g., observation of both the

lactam and the monodehydrated lactam for a given residue. Mascot analysis of the spectra

from the in-gel tryptic digest (31% sequence coverage, encompassing 13% of the Lys

residues) did not identify any modified peptides with a significant score. In contrast,

analysis of the in-gel chymotryptic digest (67% sequence, 28% Lys coverage) identified

one distinct peptide, SVTGAK280QVNY, modified at Lys280 (Table 2.2S). In this

peptide, the modified lysine residue was within the peptide and not at the C terminus,

indicating that modification blocks tryptic cleavage. Thus, based on our evaluation

criteria, we identified one lysine residue in calpain-1 as modified by iso[4]LGE2.

Figure 2.3 shows a full MS comparison of iso[4]LGE2 unmodified and modified

calpain-1 peptide “SVTGAKQVNY” with modification at K280. The mass spectrum of

the modified peptide revealed two forms of adducts, a lactam and a monodehydrated

lactam, both as doubly charged ions (Table 2.1). In contrast, the spectrum of the

unmodified peptide did not exhibit peaks for either of these modifications (Table 2.1).

Due to their hydrophobicity, modified peptides eluted later than the unmodified peptide.

41

It is likely that the loss of water to form the monodehydrated lactam occurred in the mass

spectrometer.

Figure 2.3 MS of iso[4]LGE2-modified and unmodified calpain-1 peptide

“SVTGAKQVNY” with the modification site at K280. Panel A: Detection of the doubly

charged ions m/z 699.88 and 690.88 corresponding to the lactam and monodehydrated

lactam modifications of SVTGAKQVNY. Panel B: Detection of the doubly charged ion

m/z 533.78 corresponding to the unmodified peptide. See table 2.1 for calculated

molecular weights.

Table 2.1 Molecular weight of iso[4]LGE2-modified and unmodified calpain-1 peptide

“SVTGAKQVNY”.

Peptide Sequence Formula of

MW of

ΔMass Found in

Elution

Notes

SVTGAKQVNY No Mod 1065.66 Da Yes 33.18' No Modification

SVTGAKQVNY C20H28O3 1381.20 Da 316.20 Da No ND Pyrrole

SVTGAKQVNY C20H26O2 1363.86 Da 298.20 Da No ND Pyrrole Monodehydrated

42

SVTGAKQVNY C20H28O4 1397.76 332.10 Da Yes 61.54' Lactam

SVTGAKQVNY C20H26O3 1379.76 Da 314.10 Da Yes 61.54' Lactam Monodehydrated

To unambiguously establish the site of modification, MS2 analysis was performed to

study the fragmentation patterns of the modified versus the unmodified chymotryptic

peptide. A series of fragment y-ions and b-ions was detected that identify an iso[4]LGE2

modification on the Lys residue of the calpain-1 peptide SVTGAKQVNY (Figure 2.4).

S V T G A K280(IsoLG Lactam-H2O) Q V N Y

y

b

123456789

y*(2)y(2)

y(3)b(5)

y(4)

b*(9)++, b0(9)++

b(9)++

b0(6)

b(6)

y(5)

b(7)

b0(8)

b(8)

b*(9)

b(9)

m/z

Rel

ativ

e A

bund

ance

m/z of Lys=128.09m/z of lactam dehydrated=314.19128.09 + 314.19=442.28m/z from y4 to y5=442.28

Figure 2.4 Tandem MS characterization of the m/z 690.88 doubly charged ion from a

MS scan of chymotrypsin digested iso[4]LGE2-modified calpain-1 showed a series of

fragment ions that unambiguously identify an iso[4]LGE2 modification on the lysyl

residue (K280). Asterisks denote fragment ions with a modified lysyl residue.

The iso[4]LGE2-modified chymotryptic peptides that we identified enabled

determination of the specific lysyl group that was altered in various samples of

iso[4]LGE2-modified calpain-1. MS/MS analysis consistently detected modification of

the SVTGAKQVNY chymotryptic peptide from the catalytic subunit of iso[4]LGE2-

43

calpain-1 in samples of iso[4]LG-modified calpain-1 that were generated with various

iso[4]LGE2 to protein ratios, showing that this sequence was readily accessed and

covalently altered by this γ-ketoaldehyde electrophile. Figure 2.5 shows ball-and-stick

and cartoon models that provide 3D visualization of calpain-1 using PyMOL v1.4.1. All

lysines of calpain-1 are shown as space-filling models in the left panel while only the

lysine that became incorporated into the iso[4]LGE2-modification and three active site

catalytic triad residues (Cys 115, His 272 and Asn 296) are shown in the right panel

cartoon model. The 3D model shows that the modified lysine is located at and protrudes

from the surface of calpain-1. The physical environment and chemical characteristics of

this specific lysyl residue apparently facilitates access by iso[4]LGE2 and covalent

adduction through Schiff base formation with its aldehyde group followed by cyclization

and pyrrole formation. In vivo, phospholipid derivatives containing the iso[4]LG

esterified to the sn2-hydroxyl protrude like whiskers28 from membrane bilayers and the

surface of lipoprotein particles. These isoLGs esterified to phospholipids adduct to

proteins to initially form Schiff basses and then cyclized to pyrroles. At some point,

phospholipolysis releases the free carboxyl of the isoLG.

44

Figure 2.5 Ball-and-stick and cartoon 3D model models for the catalytic subunit of

calpain-1(pdb: 2ary). All the lysines were shown as space-filling models in the left panel.

Only lysine (280) that was modified by iso[4]LGE2 and three active site amino acid

residues (Cys 115, His 272 and Asn 296) were shown in the right panel.29-31

The architecture of calpain-1 consists of a short N-terminal extension (domain I),32

the catalytic core (dIIa and dIIb), the C2-like domain III,33 and a penta-EF hand domain

IV,31 which interacts with the small subunit domains dV34 and dVI (Figure 2.1S, left

panel).35-36 Subdomain IIa and domain IIb, with a substrate-binding cleft in between,

comprises domain II, also name as minicalpain. The catalytic triad residue Cys 115 is on

subdomain IIa, whereas His 272 and Asn 296 are part of subdomain IIb. In the absence of

calcium, the distance between the catalytic Cys 115 and His 272 (10 Å) is too great to

form a functional catalytic triad. It is suggested that calcium-induced conformational

45

changes draw subdomain IIa and IIb together29, 37-3829, 37-3829, 37-3829, 37-38 (Figure 2.1S, right

panel).29, 37-38 As described above, calpain-1 loses protease activity when modified with

iso[4]LGE2. The modified lysine 280 is located on β strand 11 (amino acid sequence 274-

284) which is close to but not in the calpain-1 active site (His 272). The loss of activity

consequent to modification of this lysine may result from a conformational change

caused by the modification (Figure 2.6).

Iso[4]LGE2

Lys280

β12 β11

β13

Figure 2.6 The active sites of human minicalpain 1 are shown in stick representation and

labeled (left panel).30 Modified lysine 280 and the active site (Cys 115, His 272 and Asn

296) and related β strands are shown in the cartoon 3D model for the catalytic subunit of

calpain-1 (right panel).

2.2.3 In vitro modification of calpain-1 with iso[4]LGE2 generates cross-links.

Human serum albumin (HSA) binds ten equivalents of LGE2 within 1 minute.39 The

initial lysyl-LG adducts with proteins are also reactive, eventually undergoing

intermolecular protein-protein cross-linking, e.g., LGE2 also causes extensive

intermolecular cross-linking of ovalbumin.40

46

The time course of intermolecular cross-linking of calpain-1 was monitored upon

incubation with 2 to 460 equivalents of iso[4]LGE2. The oligomerizations were

terminated after various time intervals by the addition of excess glycine, and the

oligomers were fractionated by SDS-PAGE and Western analysis (see Figure 2.7 and

appendix Figure 2.2S-2.6S). Dimers and higher oligomers were already prominent after 1

h. Primary antibodies against the catalytic and small regulatory subunits as well as anti-

iso[4] monoclonal antibody were used for Western analysis to detect both calpain-1 and

iso[4]LGE2 modified calpain-1. All the images of the gels include the loading well. Only

the incubation with 460 equivalents of isoLG showed an appreciable accumulation of

high molecular weight oligomer in the loading well.

Use Anti-Calpain 1 catalyticsubunit primary antibody

2025

37

50

75

100

150

250

12 h1 h 3 h24 hControl

24 h

240 kDa160 kDa

320 kDa

120 kDa110 kDaCatalytic subunit--80kDa

Modification

Use Anti-Calpain 1 smallsubunit primary antibody

2025

37

50

75

100

150

250

12 h1 h 3 h24 hControl

24 h

140 kDa120 kDa110 kDa

Small Subunit--30kDa

Catalytic subunit--80kDa

Modification


Use Anti-Iso[4]LGE2 monoclonal antibody

12 h1 h 3 h 24 h2025

37

50

75

100

150

250


160 kDa240 kDa

320 kDa


Modification

Figure 2.7 Western analysis of iso[4]LGE2 (50 equivalents) modified calpain-1 after

reaction for 1h, 3h, 12h and 24h. Left panel: developed with anti-calpain-1 catalytic

subunit primary antibody. Middle panel: developed with anti-calpain-1 small subunit

primary antibody. Right panel: developed with anti-iso[4]LGE2 monoclonal primary

antibody.

47

Notably, intense bands for the catalytic subunit, its dimer and trimer at 80, 160 and

240 kDa were detected with anti-calpain-1 catalytic subunit antibody but not with the

regulatory subunit antibody. The dimer and trimer, in contrast with the monomer, were

also not prominently stained by the anti-iso[4]LG antibody suggesting that the antibody

does not detect isoLG involved in cross-linking and that the cross-linked catalytic

subunits do not have extensive additional modification that is not involved in the cross-

link. For example, with 50 equivalents of iso[4]LGE2 (Figure 2.7), Western analysis

revealed that within in 1 h, a dimer, higher oligomers and even higher multimers

appeared. By contrast, the control, calpain-1 in the absence of iso[4]LGE2, exhibited no

oligomerization after incubation for 24 h. The anti-calpain-1 catalytic subunit (80 kDa)

primary antibody detected a band at 110 kDa corresponding to a hetero dimer of one

catalytic subunit with one regulatory small subunit (30 kDa). It also detected bands at 160

kDa, 240 kDa and 320 kDa corresponding to a dimer, trimer and tetramer of the catalytic

subunit. The anti-calpain-1 small subunit primary antibody (30 kDa) also detected a band

at 110 kDa corresponding to a hetero dimer of one catalytic subunit and one small

regulatory subunit. It also revealed a band at 120 kDa corresponding to a tetramer of the

small regulatory subunit, and a band at 140 kDa corresponding to a heterotrimer

consisting of two small regulatory subunits cross-linked with one catalytic subunit. Both

the anti-catalytic subunit and small subunit primary antibodies showed some, albeit low,

cross-reactivity. The anti-iso[4]LGE2 monoclonal antibody detected virtually all oligomer

bands resulting from modification most likely due to non-crosslinked modification.

Although the molecular structure of the protein cross-link was unknown,40-41 the

48

oligomers are resistant to dissociation by SDS and heat under reducing conditions,

indicating a high probability that the cross-link involves covalent bonding.

2.2.4 Preparation of fluorescently labeled iso[4]LGE2.

The identification and quantitation of protein cross-linking have proved to be a

formidable analytical challenge in proteomics. Fluorescence detection-HPLC is an

important method for trace analysis and in combination with mass spectrometry may

provide an approach to analyze isoLG modified protein cross-linking. For this purpose,

stable fluorescently labeled iso[4]LGE2 was developed and tagged to protein through

modification. After digestion, tagged samples were subjected to LC/MS with

fluorescence detection for further analysis.

Preparation of fluorescently labeled iso[4]LGE2 2.4 was achieved in two steps

(Scheme 2.2). Iso[4]LGE2 precursor 2.1 was synthesized as described.3 (4-(N,N-

Dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole) (DBD-ED)42-44 is

a widely used fluorescence labeling reagent which emits strong fluorescence at long

wavelengths. Its 2-aminoethylamine group reacts in the presence of a condensing agent

with carboxylic acids at room temperature to yield a stable amide. The EDCI-HoBt45-49

system was chosen as condensing agent after testing triphenylphosphine (TPP), 2,2'-

Dipyridyldisulfide (DPDS)50-54 and 1,1'-Carbonyldiimidazole (CDI).55 Fluorescently

labeled iso[4]LGE2 intermediate 2.3 was prepared by treating precursor 2.1 with EDCI-

HoBt first and then adding DBD-ED at 25 °C in the dark. After overnight reaction, one

product was formed and unreacted materials could be easily removed by flash silica gel

chromatography. Stirring the acetal 2.3 in dilute acetic acid overnight followed by

49

evaporation of the solvent under high vacuum and flash chromatography accomplished

hydrolysis of the dimethyl acetal to cleanly deliver pure 2.4. MS spectra were shown in

appendix Figure 2.7S.

CO2H

O

(MeO)2HCOH

C5H11

NHCH2CH2NH2

SO2N

NO

N

CH3CH3

EDCI-HOBt

C

O

(MeO)2HCOH

C5H11

ONHCH2CH2NH

NO

N

SO2NCH3

CH3

AcOHH2O

C

O

OH

C5H11

ONHCH2CH2NH

NON

SO2NCH3

CH3

O

2.1

2.2

2.3

2.4

Scheme 2.2 Synthesis of a fluorescently labeled iso[4]LGE2 derivative.

2.2.5 GAPDH Lys3 and Lys249 are modified by fluorescently labeled isoLG.

Fluorescently labeled isoLG was first used to detect the sites of 1:1 lysine-isoLG

adducts, e.g., pyrroles, lactams, hydroxylactams and their derivatives, on a homotetramer

protein glyceraldehyde 3-phosphate dehydrogenase. Fluorescently labeled iso[4]LGE2-

treated GAPDH was trypsinyzed in-solution followed by triple quadrapole MS and Q-Tof

MS/MS analysis of tryptic peptides. Peak lists from the acquired MS/MS spectra were

analyzed with the Mascot data base search engine. Evaluation criteria for identification of

the modified peptides were the same as normal iso[4]LGE2 modified peptides. Lysine

modifications and chemical structures of lys-fluorescently labeled iso[4]LGE2 adducts

were presented in Table 2.4S and Scheme 2.1S. This analysis identified two lysine

residues in GAPDH that were modified by fluorescently labeled iso[4]LGE2 as a

monodehydrated lactam and a bisdehydrated hydroxylactam. MS/MS characterization of

50

the doubly charged ion m/z 620.86 (LEKAAK) and triply charged ion m/z 537.96

(VKVGVNGFGR) showed a series of fragment ions sufficient to unambiguously identify

a fluorescently labeled iso[4]LGE2 modification on the GAPDH lysyl residues K3 and

K249 (Table 2.2 and Table 2.5S).

Table 2.2 Tryptic peptides from GAPDH modified by fluorescently labeled iso[4]LGE2.

Sequence Observed Mr(expt) Ion score Variable Modification

LEK (-C

30H

39N

5O

5S)AAK 620.8596(2+) 1239.7046 18 Lactam Monodehydrated

VK (-C

30H

37N

5O

5S)VGVNGFGR 537.9638(3+) 1610.8696 23 Hydroxylactam Bisdehydrated

MS/MS analysis consistently detected modification on the LEKAAK and

VKVGVNGFGR sequences of fluorescently labeled iso[4]LGE2-GAPDH in preparations

generated with various equivalents of fluorescently labeled iso[4]LGE2, implying that

these sequences were readily accessed and covalently altered by this aldehyde

electrophile. Figure 2.8 showed ball-and-stick and cartoon models providing 3D

visualization of GAPDH using PyMOL v1.4.1. All lysines of GAPDH are shown as

space-filling models in the left panel while only the lysines that becoming adducted by

fluorescently labeled iso[4]LGE2 and active site are shown in the cartoon model in the

right panel. The 3D model shows that these lysines are located at and protrude from the

surface of GAPDH. The physical environment and chemical characteristics of these

specific lysyl residues probably facilitated access by iso[4]LGE2 and covalent adduction

through Schiff base formation with its terminal aldehyde group leading to pyrrole

formation. This is similar to the preference noted above for isoLG modification of

calpain-1.

51

Figure 2.8 A ball-and-stick and cartoon 3D model structure for catalytic subunit of

GAPDH (pdb: 1j0x). All the lysines were shown as space-filling models in the left panel.

Only lysines that became modified by fluorescently labeled iso[4]LGE2 and active site

(Cys 149) were shown in the right panel.55-56

2.2.6 In vitro modification of GAPDH with fluorescently labeled iso[4]LGE2 forms

cross-links.

The time course of intermolecular cross-linking of GAPDH upon incubation with 10

equivalents of fluorescently labeled iso[4]LGE2 was determined by quenching the

oligomerization after various time intervals by the addition of excess glycine, and

fractionating the oligomers by SDS-PAGE. Only the 24 h incubation showed an

accumulation of high molecular weight oligomer in the loading well (Figure 2.9). Within

1 h, a dimer, trimer and even higher multimers appeared. By contrast, GAPDH in the

absence of fluorescently labeled iso[4]LGE2 exhibited little or no oligomerization after

incubation for 24 h. The traces of oligomers that were barely detectable in the control

probably represent minor alternative oligomerization mechanisms.

52

1h 4h2h 8h 24h 24 hControl

monomer

tetramer

trimerdimer

oligomer

263443567295

130

170

Modification

Figure 2.9 Coomassie blue staining of 10 equivalents fluorescently labeled iso[4]LGE2

modified GAPDH after incubation for various times prior to addition of excess glycine.

2.2.7 Pilot studies on cross-linking during modification of GAPDH with

fluorescently labeled iso[4]LGE2.

2.2.7.1 Amino acid analysis GAPDH dimer, trimer and tetramer formation upon

modification with fluorescently labeled iso[4]LGE2.

Amino acid analysis was performed to quantify the percentage of modified GAPDH

cross-linked as dimer, trimer and tetramer contained in a sample. The identity and

quantity of each amino acid was determined by comparing retention time and

chromatographic peak areas detected from the sample with those of a calibration file that

was generated from an amino acid standard mixture (standard H, Pierce).

Dimer, trimer and tetramer fractions of fluorescently labeled iso[4]LGE2 modified

GAPDH were cut from a gel (Figure 2.9) and individually trypsin in gel-digested.

53

AccQ·TagTM amino acid analysis was used to quantify each cross-linked fraction in a

particular sample of GAPDH. First the tryptic peptides from the sample were hydrolyzed

into free amino acids under HCl vapor. Then the amino acids were derivatized using a

Waters AccQ·TagTM ultra derivatization kit. AccQ·TagTM ultra reagent reacts rapidly

with primary amino groups to yield highly stable urea. The structure of the UV active

derivatizing group is the same for all amino acids. Excess reagent hydrolyzes to yield 6-

aminoquinoline (AMQ), a non-interfering by-product.

N

HN O

NO

O

O

NHR1 R2Amino Acid

t 1/2<<1s N

HN N

O

R1

R2

Derivalized Amino Acid

+ N

O

O

HO

N-HydroxysuccinimideH2O t 1/2<<15s

N

NH2+ N

O

O

HO

N-Hydroxysuccinimide

+ CO2

6-aminoquinoline

Scheme 2.3 AccQ·TagTM derivatization.65-66

Finally, the derivatized hydrolysate was chromatographically analyzed. The identity

and quantity of each amino acid was determined by comparing retention time and

chromatographic peak areas with the peak area of the quantitative standard amino acids

(Standard H, 50 pmol, appendix Figure 2.8S). The percent of total protein sample that

corresponds to the fraction analyzed was determined by dividing the amount determined

by the quantitative amino acid analysis of the fraction by the total amount of protein

(GAPDH after 24 h modification and for SDS analysis) in the sample times 100. After 24

h incubation, about 2.5%, 2.5% and 1.5% of GAPDH was present in the sample in the

form of dimer, trimer and tetramer, respectively (appendix Table 2.6S).

54

2.2.7.2 LC/MSMS analysis of fluorescently tagged tryptic peptides

A combination of fluorescence detection-HPLC and mass spectrometry was applied

to analysis of tryptic peptides from an isoLG-modified protein (GAPDH) in an effort to

identify isoLG-cross-linked peptides. To enable detection and facilitate isolation of

isoLG-modified peptides, a fluorescent label was incorporated into the isoLG. Thus,

homo-tetrameric protein GAPDH was modified with stable fluorescently labeled

iso[4]LGE2, trypsinized, and the tryptic peptides were first separated on a reverse phase

column, fractions detected with an Acquity® fluorescence (FLR) detector were analyzed

with a triple quadrupole electrospray mass spectrometer (Applied Biosystems Inc.).

Fluorescence channels of tryptophan peptides (three digested peptides containing

tryptophan) and iso[4]LGE2 tag were both monitored. The fluorescence signal of

tryptophan peptides (280 nm for excitation, 340 nm for emission) and mass spectrometry

(ESI-triple quadrupole) were employed to evaluate the chromatographic separation of

peptides on various LC columns. A column was identified that gave sharp peaks. Then

using that column, the fluorescence signal of iso[4]LGE2 tagged peptides (450 nm for

excitation, 560 nm for emission) was monitored to detect the iso[4]LGE2 modified

peptides (see appendix Figure 2.9S and 2.10S). Peptides with iso[4]LGE2 tag

fluorescence signals were collected as one minute fractions and quickly dried using a

high-speed vacuum evaporator and stored at -20 °C until used. These peptides were re-

suspended in 0.1% formic acid/2% acetonitrile and further analyzed by LC-MS/MS using

a quadrupole-time of flight (QTof2) mass spectrometer. To identify 1:1 isoLG-peptide

adducts, peak lists from the acquired MS/MS spectra were analyzed with the Mascot data

base search engine.

55

To identify 2:1 isoLG-peptide aminal cross-links (Scheme 2.4), peptides with

fluorescence signal were collected as one minute fractions that were analyzed by MS

analysis on QTOF. The masses of all possible tryptic GAPDH peptides with one missing

cleavage were calculated (Table 2.7S). The masses of cross-linked peptide [M] were then

calculated by summing the mass of peptide #1 + mass of peptide #2 + 601.2934 (the mass

increment for the cross-link, Scheme 2.4). The exact masses calculated for the [M+H]+,

[M+2H]2+, [M+3H]3+, [M+4H]4+ ions are shown in Table 2.8S. Since the peptides

“LEKAAK” and “MVKVGVNGFGR” were found to form lysine 1:1 adducts, we

postulated that these two peptides are particularly reactive toward iso[4]LGE2.

Consequently, we considered them to be high probability candidates for forming aminal

cross-links. The molecular weights of aminal cross-links between “MVKVGVNGFGR”

+ “MVKVGVNGFGR”, “LEKAAK”+ “LEKAAK”, “LEKAAK”+ “MVKVGVNGFGR”

for ions from +1 to +4 charges were calculated and compared with all precursor ions

exacted from QTOF MS of each HPLC fraction.

N CC5H11

NHCH2CH2NHO

OH

OH

NH

NO

N

SO2Npeptide #1

peptide #2

= 604. 3169

Scheme 2.4 Chemical structure of peptides aminal cross-link. Mass of cross-linked

peptide = mass of peptide #1 + mass of peptide #2 + 601.2935 (= 604.3169 – 3.0234).

An observed ion of mass 640.9626 corresponds approximately with the m/z 640.

3732 expected for a +3 charged cross-link of the peptide “LEKAAK”+ “LEKAAK”. To

56

test the hypothesis that this ion corresponds to the above mentioned ion, MS2 daughter

ions were examined for various expected fragmentation involving loss of b2 (243), a2

(215) or “AK” (199), “AAK” (270). However, none of the expected fragments were

detected. Therefore, it is unlikely that the parent ion at 640.9626 corresponds to the

proposed cross-link (Figure 2.11S).

To supplement the approach described above, in future studies, modification of

proteins with a 1:1 mixture of d6-iso[4]LGE2 and d0-iso[4]LGE2, can provide additional

evidence that will facilitate the detection and characterization of protein cross-links. If

peptides were modified by both d6-iso[4]LGE2 and d0-iso[4]LGE2, the observation of

characteristic pairs of peaks would provide convincing evidence for the presence of an

isoLG in the ion being observed. The pair of peaks would be separated by +6 amu for

singly charged ions, +3 amu for doubly charged ions, and +2 amu for triply charged ions.

57

2.3 Conclusions


aggregation. The sites of 1:1 adduct formation and the types and extent of cross-linking

between subunits of the multi subunit proteins calpain-1 and glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) by iso[4]LGE2 were characterized mass spectroscopically and

by SDS-PAGE. Calpain-1 lost its protease activity with t1/2 = ~40 min upon incubation

with 10 equivalents of iso[4]LGE2, and as little as 2 equivalents was almost as effective.

This suggests that modification of one or two lysyl residues suffices to abolish activity.

LC-MS/MS analysis established that Lys280 is a target of isoLG adduction to calpain-1.

Since this lysine residue is not located in the active site, a conformational change induced

by the modification may account for the loss of activity. Covalently cross-linked hetero

and homo oligomers of calpain-1 catalytic and regulatory subunits were detected by SDS

PAGE. The dimer and trimer, in contrast with the monomer, of the catalytic subunit were

prominently stained by an antibody against the catalytic subunit but not by an antibody

against the regulatory subunit or by the anti-iso[4]LG antibody suggesting that the

iso[4]LG antibody does not detect isoLG involved in cross-linking and that the cross-

linked catalytic subunits do not have extensive additional modification that is not

involved in the cross-link.

The amounts of dimer, trimer and tetramer forms of GAPDH produced after 24 h

incubation with 10 equivalents of fluorescently labeled iso[4]LGE2 was determined by

amino acid analysis to be about 2.5%, 2.5% and 1.5% respectively. LC-MS/MS analysis

established that Lys3 and Lys249 are targets of fluorescently labeled iso[4]LGE2

adduction to GAPDH. These lysine residues are located in the tryptic peptides

58

“LEKAAK” and “VKVGVNGFGR”. Since the peptides “LEKAAK” and

“MVKVGVNGFGR” were found to form lysine 1:1 adducts, we postulated that these

two peptides are presumably particularly reactive toward iso[4]LGE2. Consequently, we

considered them to be high probability candidates for forming aminal cross-links.

However, no ions were detected that could be confirmed by MS/MS analysis to

correspond to these cross-linked peptides.

59

2.4 Experimental Procedures

2.4.1 General methods. All chemicals used were high purity analytical grade. The

following commercially available materials were used as received: 4-(N,N-

dimethylaminosulfonyl)-7-(2-aminoethylamino)-2,1,3-benzoxadiazole (DBD-ED) and

2,2'-dipyridyldisulfide (DPDS) were from TCI America (Portland, OR). Calpain-1 (from

human erythrocytes) was from Calbiochem (La Jolla, CA). 1-Hydroxybenzotriazole

(HoBt), iodoacetamide, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride

(EDCI), 1,1'-carbonyldiimidazole (CDI), and GAPDH (from rabbit muscle) were from

Sigma (St. Louis, MO). Dithiothreitol was from Denville Scientific (South Plainfield NJ).

Chymotrypsin and trypsin were from Promega (Madison, WI). Sodium acetate,

ammonium bicarbonate, disodium hydrogen phosphate and monosodium phosphate were

from Fisher Scientific Co. Iso[4]LGE2 was prepared by Dr. Jim Laird and iso[4]LGE2-

FluoTag was prepared as discussed below.

Chromatography was performed with ACS grade solvent. Thin layer chromatography

(TLC) was performed on glass plates precoated with silica gel (Kieselgel 60 F254, E.

Merck, Darmstadt, West Germany). Rf values are quoted for plates of thickness 0.25 mm.

The plates were visualized by viewing the developed plates under short or long

wavelength UV light, iodine, or by heating the plates after dipping in 20% solution of

phosphomolybdic acid in ethanol. Flash chromatography was performed according to

established methods57 on ICN SiliTech 32-63 D 60A silica gel from ICN Biomedicals

Gmbh, Eschwege, Germany.

60

Software: DeepView from Swiss-PdbViewer (http://spdbv.vital-it.ch/), SWISS-

MODEL Workspace (http://swissmodel.expasy.org/) and PyMOL v1.4.1

(http://pymol.org/)

2.4.2 Synthesis of fluorescently labeled iso[4]LGE2. HOBt (1.48 mg, 11 µmol in 40 µl

DMF), EDCI (2.12 mg, 11 µmol in 56 µl CH2Cl2), and precursor 2.1 (4 mg, 10 µmol in

50 µl CH2Cl2) were stirred at room temperature for 2.5 h. A solution of DBD-ED (11

µmol, 2.84 mg) dissolved in 140 µl of acetonitrile was added to the reaction mixture.

After overnight reaction in the dark, TLC showed only one major product formed and

complete disappearance of precursor 2.1. The reaction mixture was concentrated and the

residue was purified by flash chromatography on a silica gel column (ethyl acetate:

methanol=20:1, TLC: Rf = 0.7) to give the fluorescently tagged iso[4]LGE2

dimethylacetal derivative 2.3 (4.8 mg, 7 µmol, yellow oil). ESI-MS: m/z calcd for

C32H51N5O8S (M), 665.35; found, 664.13 (M-H) 700.00 (M-H+Cl) for negative mode,

and 688.33 (M+Na) for positive mode.

Hydrolysis of the dimethyl acetal in 2.3 was accomplished by dissolving it (4 mg, 6

µmol) in 3:1 acetic acid/water (1.3 mL) and incubating overnight at room temperature in

the dark, followed by rotary evaporation of solvents. The residue was purified by flash

chromatography on a silica gel column (ethyl acetate: methanol=40:1, TLC: Rf = 0.6) and

placing under high vacuum through a dry ice-acetone cooled trap for 2 days to give

compound 2.4. (2.3 mg, 3.8 µmol, yellow solid). ESI-MS: m/z calcd for C30H45N5O7S

(M), 601.28; found, 600.13 (M-H), 635.87 (M-H+Cl) for negative mode, and 624.20

(M+Na) for positive mode.

61

http://spdbv.vital-it.ch/

http://swissmodel.expasy.org/

http://pymol.org/

2.4.3 In vitro modification of calpain-1 by iso[4]LGE2. Calpain-1 was modified with

10 equivalents of iso[4]LGE2. Calpain-1 (100 µg, 8.9 nmol) in 190 µl of a solution

containing 20 mM imidazole, 5 mM β–mercaptoethanol, 1 mM EDTA, 1 mM EGTA, 30 %

glycerol, pH 6.8 was mixed with 310 μl of sodium acetate solution (10 mM, pH 6.8).

First 11 µl of iso[4]LGE2 stock (1 µg/µl in methanol, 11 µg, 30 nmol) was added to

calpain-1 and the mixture reacted on a shaker (IKA MTS 2/4 digital microtiter shaker,

300 rpm) for 40 min. Then another 11 µl of iso[4]LGE2 stock (1 µg/µl, 11 µg, 30 nmol)

was added and reacted for 40 min, then the last 11 µl of iso[4]LGE2 stock (1 µg/µl, 11 µg,

30 nmol) was added and reacted for 40 min. The reaction mixture (total 533 µl) was

quenched by addition of glycine (2 µl, 100 mM, 178 nmol, 2 molar equivalent to

iso[4]LGE2) with brief mixing. The mixture was then flash-frozen in liquid nitrogen and

stored at -80 °C until analysis by MS.

Modified calpain-1 (20 µg) was subjected to 4-12% SDS-PAGE. The gel was stained

with Coomassie blue, and a region corresponding to proteins with molecular mass

between 80-90 and 30-40 kDa was excised respectively. Destained, reduced in 20 mM

DTT for 30 min at room temperature, and alkylated in 100 mM iodoacetamide in the dark

for 30 min at room temperature. For LC/MSMS analysis, the protein was first in gel

digested with chymotrypsin. Chymotrypsin stock solution was prepared by dissolving

chymotrypsin (25 µg) in NH4HCO3 (1000 µl 50 mM), to provide a final concentration of

25 µg/ml. Chymotrypsin solution (16 µl, 0.4 µg) was added to modified calpain (20 µg)

for 22 h at 37 °C using a protease to calpain-1 ratio of 1:50 (w/w) in 50 mM NH4HCO3.

Peptides were extracted with 50% acetonitrile and 5% formic acid, dried in a vacuum

concentrator, and stored at -20 °C. Peptide separations were performed on an Ultimate

62

3000 LC system with a C18 Acclaim PepMap 100 column (0.075 x 150 mm, Dionex,

Sunnyvale, CA). Peptides were eluted over a 50-min gradient from 0 to 80% acetonitrile

in water, containing 0.1% formic acid, at a flow rate of 300 nl/min. The column effluent

was continuously directed into the nanospray source of the mass spectrometer, a hybrid

Fourier transform ion cyclotron resonance (FTICR)/linear ion trap mass spectrometer

(LTQ FT Ultra, Thermo Scientific, West Palm Beach, FL). The following parameters

were used for all acquisition methods on the LTQ FT Ultra MS: an ion spray voltage of

2400 V and an interface capillary heating temperature of 200 °C. Full mass spectra were

acquired from the FTICR, and the tandem mass spectra (MS/MS) of the eight most

intense ions were recorded by the linear ion trap in data-dependent mode with normalized

collision energy of 35 eV, isolation width of 2.5 Da, and activation Q of 0.25.

2.4.4 In vitro modification of GAPDH by fluorescently labeled iso[4]LGE2. GAPDH

was modified with 10 equivalents of fluorescently labeled iso[4]LGE2. Fluorescently

labeled iso[4]LGE2 (168 µg, 280 nmol) (21 µl of 8 mg/ml in methanol) was added to into

GAPDH (28 nmol) (1 mg in 479 µl of 50 mM sodium phosphorus buffer, pH 7.0) and the

mixture was under air on an IKA MTS 2/4 digital microtiter shaker at 300 rpm for a total

8 h at 25 °C. Then the reaction mixture (total 500 µl) was quenched by addition of

glycine (6 µl, 100 mM, 560 nmol, 2 molar equivalent to fluorescently labeled iso[4]LGE2)

with brief mixing. The mixture was flash-frozen in liquid nitrogen and stored at -80 °C

for further analysis by MS and amino acid analysis.

For LC/MSMS analysis, in solution digestion was performed using trypsin. The

protein was reduced with 20 mM DTT for 30 min at room temperature, and alkylated in

63

200 mM iodoacetamide in the dark for 1 h at room temperature. The protein was then

digested by trypsin (20 µg) for 24 h at 37 °C with a protease to GAPDH ratio of 1:50

(w/w) in 50 mM of NEM·OAc (pH 8.6). Peptides were dried in a vacuum concentrator,

and the residue stored at -20 °C.

The tryptic peptides were first separated on a reverse phase column (2.1x100 mm i.d.

C18 1.8 µm column from Waters Acquity UPLC HSS). Chromatography was carried out

by elution with a linear gradient (elute A, 0.1% formic acid/H2O; elute B, 0.1% formic

acid/ACN). The sample was loaded onto the column with 98% A and 2% B at a flow rate

of 70 µl/min for 10 min. For the analysis, the gradient was as follows: 2-10% B over 5

min, to 70 % within 30 min at a flow rate of 70 µl/min, to 98 % within 0.1 min and hold

for 5 min at a flow rate of 140 µl/min.

The analyses of digested peptides were performed by Acquity® fluorescence (FLR)

detector and API-3000 triple quadrapole electrospray mass spectrometer (Applied

Biosystems Inc.) operated in the positive ion mode and a MS scan of m/z 220-2000.

Fluorescence channels of tryptophan peptides (three digested peptides containing

tryptophan) and iso[4]LGE2 tag were both monitored. The fluorescence of tryptophan

peptides (280 nm for excitation, 340 nm for emission) and mass spectrometry were

monitored to confirm the chromatographic separation. The fluorescence signal of

iso[4]LGE2 tag (450 nm for excitation, 560 nm for emission) was monitored to confirm

the elution time of fluorescently labeled iso[4]LGE2 modified peptides. Peptides with

iso[4]LGE2 tag fluorescence signals were collected as one minute fraction, quickly dried

using a high-speed vacuum evaporator and then stored at -20 °C until for further analysis.

64

The tryptic peptides with fluorescence signals were re-suspended in 0.1% formic

acid/2% acetonitrile and further analyzed by LC-MS/MS using a quadrupole-time of

flight (QTof2) mass spectrometer equipped with a CapLC system (Waters, Millford, MA),

ProteinLynx Global Server acquisition and processing software. Peptide digests were

trapped on a precolumn (0.3 x 1mm, 5µC18, LC Packing) with 0.1% formic acid in 2%

acetonitrile as loading solvent then eluted onto a capillary column (PicoFrit 0.050 x 75

mm, 15µ tip ID; New Objective Inc., Woburn, MA) over a 60-min gradient from 0 to 80%

acetonitrile in water, containing 0.1% formic acid, at a flow rate of 250 nl/min. The

column effluent was continuously directed into the nanospray source of the mass

spectrometer which was operated in standard MS/MS switching mode with the three most

intense ions in each survey scan subjected to MS/MS analysis. Protein identifications

from MS/MS data utilized ProteinLynxTM Global Server (Waters Corporation) and

Mascot (Matrix Science) search engines and the Swiss-Protein and NCBI protein

sequence databases.

2.4.5 Electrophoresis. According to a published protocol,58 modified proteins (~ 5-10 µg)

were suspended in Laemmli sample buffer,59 fractionated on a 4-12% SDS-PAGE, and

then staind with coomassie blue (Bio-Rad coomassie stain #161-0786) or silver stain (GE

Healthcare, PlusOne silver staining kit, protein, instructions 71-7177-00AL). The bands

were quantified using a GS-710 scanner and QuantityOne® software from BioradTM

(Hercules, CA).

65

http://www.uvic.ca/medsci/assets/docs/research-facilities/quantity-one-user-guidel.pdf

2.4.6 Western analysis. According to published protocols,58, 60 modified proteins (~ 5-10

µg) were suspended Laemmli sample buffer,59 fractionated on a 4-12% SDS-PAGE, and

electroblotted onto a polynivyl difluoride (PVDF) membrane (Millipore, Bedford, MA).

The PVDF membranes were treated with Odyssey blocking buffer (Li-Cor Cat# 927-

40000) and probed using various antibodies: anti-calpain-1 small subunit (Fisher

Cat#01674303) mAb, anti-calpain-1 catalytic subunit (Fisher Cat#01674304) mAb, anti-

iso[4]LGE2 mAb (see Chapter 5). Goat anti-mouse IRDye® 680 (Odyssey Cat#926-

32220) was used as secondary antibody. The immunoreactive bands were detected and

quantified using a Li-Cor infrared imaging system.

2.4.7 Calpain-1 activity assay. Calpain-1 assay was performed on aliquots of modified

isoLG protein using a calpain activity assay kit (Biovision Inc., Mountainview, CA)

following recommended protocols. The fluorometric assay was based on detecting the

cleavage of a calpain substrate, Ac-LLY-AFC, that fluoresces at 505 nm, whereas the

cleaved substrate fluoresces at 400 nm. Thus, calpain-1 (26 μl of 2.286 μM, 6.6 μg,

0.0595 nmol) was added to iso[4]LGE2 (0.21 μg 0.595 nmol) in 74 μl of aqueous sodium

acetate (10 mM), mixed well and reacted under air on shaker (IKA MTS 2/4 digital

microtiter shaker, 300 rpm) at 25 °C. At each time point (3, 10, 30 and 90 min), one

aliquot (25 μl) was taken and quenched with 1.2 μl 1 mM glycine (1.2 nmol) before

activity assay. The aliquot (26.2 μl) was added extraction buffer (58.8 μl), 10x reaction

buffer (Ready-to-Use reagent from Biovision calpain activity assay kit) (10 μl) and

calpain substrate (5 μl). Then the mixture was incubated at 37 °C for 1 hour in the dark

and fluorescence was detected using Spectra Max Gemini spectrofluorometer (excitation

66


400 nm, emission 505 nm) (Molecular Devices, Sunnyvale, CA). Negative control was

performed the same method with calpain inhibitor Z-LLY-FMK from Biovision calpain

activity assay kit.

2.4.8 MS/MS identification of isoLG-modified sites in calpain-1 and GAPDH.

Peptides were identified from LTQ FT Ultra MS/MS and quadrupole-time of flight (Q-

Tof-2) experimental data by generating peak lists with Mascot Daemon and submitting

these to the Mascot search engine, version 2.3.0 (Matrix Science, Boston). S-

Carbamidomethylation of cysteine was set as a fixed modification, whereas oxidation of

methionine (methionine sulfoxide) was set as a variable modification. Formulas

corresponding to the various oxidation and dehydration states of iso[4]LGE2 adducts61

and fluorescently labeled iso[4]LGE2 adducts were manually entered into the Mascot

search parameters and selected as variable modifications for lysine (appendix Table 2.1S

and 2,4S, structures shown in Scheme 2.1S). Mass tolerances were ±15 ppm for precursor

ions and ± 0.8 Da for fragment ions. One missed cleavage site was allowed for trypsin

(cleaves at residues Lys and Arg but not at Pro) and three missed cleavage sites were

allowed for chymotrypsin (cleaves at Phe, Leu, Trp, and Tyr but not at Pro). Searches

were restricted to a sequence data base containing only human calpain-1 and rabbit

GAPDH because purified protein was used. Only peptides with a significane score

greater than 15 (p<0.05) according to Mascot’s scoring algorithm were considered.

2.4.9 Amino acid analysis. IsoLG modified GAPDH (400 µg) was subjected to 4-12%

SDS-PAGE. The gel was stained with Coomassie blue, and a region corresponding to

67

proteins with molecular mass between 70-80, 110-120 and 140-160 kDa were excised.

Each excised region was destained, reduced in 20 mM DTT for 30 min at room

temperature, and then alkylated in 200 mM iodoacetamide in the dark for 1 h at room

temperature. The protein was then digested in gel with trypsin (8 µg) for 24 h at 37 °C

with a protease to GAPDH ratio of 1:50 (w/w) in 50 mM of NEM·OAc (pH 8.6).

Peptides were extracted from the gel in 0.1%TFA/20%ACN, dried in a vacuum

concentrator, and stored at -20 °C for later analysis. AccQ·TagTM amino acid analysis

was used to quantify cross-linked GAPDH. The trypsin digested peptides were

hydrolyzed to amino acids with HCl, then with derivatized AccQ·Tag reagent and

analyzed chromatographically as described below.

2.4.9.1 Vapor phase HCl Hydrolysis: Digested dimer, trimer and tetramer peptides

were treated separately. Each sample was added 50 µl 0.1%TFA/20%ACN and then 150

µl 0.1%TFA. 20 µl aliquot was taken out from each tube and hydrolyzed to free amino

acids under HCl vapor. Peptide solutions (20 µl) were transferred to 6×50 mm hydrolysis

tubes and dried under vacuum. After drying, the glass tubes were placed into a screw-cap

vial containing 300 μl of 6M HCl and a few crystals of phenol (~1-2 mg). The vial was

evacuated briefly then flushed with argon and this alternating process was repeated three

times. The vial was finally sealed under vacuum and was heated at 150 °C in an oven for

1 h. After hydrolysis, sample tubes were dried using a high-speed vacuum evaporator and

stored at -20 °C until derivatization.

2.4.9.2 AccQ·Tag Derivatization and chromatography: AccQ·TagTM ultra

derivatization kit (Waters) was used. AccQ·TagTM ultra reagent reacts rapidly with

primary amino groups to yield highly stable urea. The structure of the derivatizing group

68

was the same for all amino acids.62-63 Peptide hydrolysates were transferred to reaction

vials and AccQ·TagTM borate buffer (30 µl) was added to adjust the pH to above 9.5.

After borate buffer was mixed well with the peptide hydrolysates by vortexing. To a 24

µl aliquot was added AccQ·TagTM reagent (6 µl, Ready-to-Use reagent from Waters

AccQ·TagTM ultra derivatization kit). The contents of the vial were mixed by vortexing

and then heated for 10 min at 55 °C. Then deionized water (30 µl) was added and the

mixture was vortexed. All 60 µl of the sample solution was transferred to a UPLC sample

vial for testing. The amino acid standard was prepared in the same manner.

The derivatized samples and H-standard were analyzed with a Waters Acquity ultra

performance LC system using 2 µl injections for optimum resolution and sensitivity (100

x 2.1 mm column). For the separation, sodium acetate (140 mM) containing 17 mM

triethylamine (pH 5) was used as buffer A and 60 % acetonitrile/H2O as buffer B. The

gradient was as follows with a flow rate of 700 μl/min: time 0-0.54 min, 0.1% B; time

5.74 min, 9.1% B; time 7.74 min, 21.2 % B; time 8.04 min, 59.6% B and time 8.05 min,

90% B. AccQ·Tag amino acids were detected with the Acquity UPLC tunable UV

detector at 260 nm.

2.4.9.3 Amino acid analysis and data interpretation: Amino acid analysis was

performed to quantify the concentrations of digested dimer, trimer and tetramer peptides. The

amount of dimer, trimer and tetramer were determined by (i) dividing the amount of each

individual residue by the known residue value, (ii) calculating a mean amount analyzed,

(iii) discarding all individual values that deviated more than 15% from the mean, and (iv)

recalculating the mean amount analyzed from the remaining relevant residues.64 The

identity and quantity of each amino acid was established by comparing retention time and

69

chromatographic peak areas with the peak area of a quantitative standard mixture of amino

acids (Standard H, 50 pmol) that was derivatized and analyzed the same way as the

digested peptides with the same amount of reagents and instruments.

Standard response (standard H) per pmol for each amino acid is equal to standard

signal (HPLC peak area) divided by pmol amino acid for analysis. Sample signal (peak

area of GAPDH derivatized dimer sample) divided by the standard response will get

analyzed pmol of each amino acid. Then analyzed sample pmol of each amino acid is

divided by the known number of amino acid per molecule protein (GAPDH) to obtain

pmol protein. Then the mean pmol protein (within 15% deviation) is calculated and

divided by sample dilution ratio to get pmol of the derivatized protein (GAPDH dimer).

Speed vac the extraction buffer, each sample was dissolved in 50 µl 0.1% TFA, 20% ACN, then added 150 µl 0.1% TFA. Total volume of each sample was 200 µl

Running gel with 400 µg (11200 pmol) of modified GAPDH (24 h)

Cut and accumulate all the dimer, trimer, tetramer bands for digestion

From each 200 µl, take out 20 µl for amino acid analysis

Dimer1.34 pmol/µl (0.05 µg/µl)

total: 0.05 µg/µl× 200 µl=10 µg 1.34 pmol/µl× 200 µl=268 pmol

modified percentage: 10µg/400µg=2.5%

Trimer1.5 pmol/ul (0.05 µg/µll)


percentage: 10µg/400µg=2.5%

Tetramer0.93 pmol/ul (0.03 µg/µl)


percentage: 6µg/400µg=1.5%

70

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protects the enzyme against inactivation by disulfiram. Biochem J 183 (3), 751-753.

51. Loader, C.; Anderson, H. J. (1971) Pyrrole Chemistry. Part XI. Some Reactions

of the Pyrrole Grignard Reagent with Alkyl Carbonates and Alkyl Thiocarbonates. Can J

Chem 49 (1), 45-48.

52. Nicolaou, K. C.; Claremon, D. A.; Papahatjis, D. P. (1981) A mild method for the

synthesis of 2-ketopyrroles from carboxylic-acids. Tetrahedron Lett 22 (46), 4647-4650.

53. Nakahara, Y.; Fujita, A.; Ogawa, T. (1985) An efficient synthesis of 2-

acylpyrroles. Agric Biol Chem 49 (5), 1491-1495.

54. Min, J. Z.; Toyo'oka, T.; Kawanishi, H.; Fukushima, T.; Kato, M. (2005) Novel

fluorescent asparaginyl-N-acetyl-D-glucosamines (Asn-GlcNAc) for the resolution of

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oligosaccharides in glycopeptides, based on enzyme transglycosylation reaction. Anal

Chim Acta 550 (1-2), 173-181.

55. Cowan-Jacob, S. W.; Kaufmann, M.; Anselmo, A. N.; Stark, W.; Grutter, M. G.

(2003) Structure of rabbit-muscle glyceraldehyde-3-phosphate dehydrogenase. Acta

Crystallogr Sect D-Biol Crystallogr 59, 2218-2227.

56. Nakajima, H.; Amano, W.; Fujita, A.; Fukuhara, A.; Azuma, Y. T.; Hata, F.; Inui,

T.; Takeuchi, T. (2007) The active site cysteine of the proapoptotic protein

glyceraldehyde-3-phosphate dehydrogenase is essential in oxidative stress-induced

aggregation and cell death. J Biol Chem 282 (36), 26562-26574.

57. Still, W. C.; Kahn, M.; Mitra, A. (1978) Rapid chromatographic technique for

preparative separations with moderate resolution. J Org Chem 43 (14), 2923-2925.

58. Crabb, J. W.; Miyagi, M.; Gu, X. R.; Shadrach, K.; West, K. A.; Sakaguchi, H.;

Kamei, M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon, R. G.; Hollyfield, J. G. (2002)

Drusen proteome analysis: An approach to the etiology of age-related macular


59. Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of head of

bacteriophage-T4. Nature 227 (5259), 680-685.

60. Bhattacharya, S. K.; Rockwood, E. J.; Smith, S. D.; Bonilha, V. L.; Crabb, J. S.;

Kuchtey, R. W.; Robertson, N. G.; Peachey, N. S.; Morton, C. C.; Crabb, J. W. (2005)

Proteomics reveal cochlin deposits associated with glaucomatous trabecular meshwork. J

Biol Chem 280 (7), 6080-6084.

61. Brame, C. J.; Salomon, R. G.; Morrow, J. D.; Roberts, L. J. (1999) Identification

of extremely reactive gamma-ketoaldehydes (isolevuglandins) as products of the

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isoprostane pathway and characterization of their lysyl protein adducts. J Biol Chem 274

(19), 13139-13146.

62. Cohen, S. A.; Michaud, D. P. (1993) Synthesis of a fluorescent derivatizing

reagent, 6-aminoquinolyl-n-hydroxysuccinimidyl carbamate, and its application for the

analysis of hydrolysate amino-acids via high-performance liquid-chromatography. Anal

Biochem 211 (2), 279-287.

63. Ward, M. (1996) Amino acid analysis using precolumn derivatization with 6-

aminoquinolyl-n-hydroxysuccinimidyl carbamate. The protein protocols handbook, 461-

465.

64. Crabb, J. W.; Ericsson, L.; Atherton, D.; Smith, A. J.; Kutny, R., A collaborative

amino acid analysis study from the association of biomolecular resource facilities. In

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Press, Inc, 1990; pp 49-61.

79

CHAPTER 3

DETECTION AND CHARACTERIZATION OF THE PRODUCTS

FROM THE REACTION OF ISO[4]LGE2 WITH PEPTIDES: N-

ACETYL-GLY-LYS-OMe AND AMYLOID “EVHHQKL”

3.1 Background

3.1.1 Purpose

Levuglandins (LGs) and isolevuglandins (isoLGs) react with protein lysyl residues to

form covalent adducts that include pyrroles and their more oxidized derivatives, lactams

and hydroxylactams, as well as protein-protein cross-links. Two alternative structures

have been proposed for the protein-protein cross-links, a pyrrole-pyrrole dimer derived

from two molecules of protein and two molecules of LG/isoLG or an aminal derived

from two molecules of protein and one molecule of LG/isoLG. In the study, to obtain

direct evidence for the molecular structure of lysyl-LG/isoLG cross-links, two simple

model systems were investigated, the reaction iso[4]LGE2 with N-acetylglycyllysine

methyl ester and with the β-amyloid (11-17) peptide EVHHQKL.

3.1.2 Isolevuglandin modification of proteins

A variety of products are formed through oxidation of fatty acids by enzymatic or free

radical induced oxidation pathways. The enzymatic oxidation of arachidonic acid (AA)

by cyclooxygenase (COX) leads to the formation of levuglandins, whereas oxidation of

phosphoesters of arachidonic acid leads to the production of isolevuglandins (isoLGs).1-3

Iso[4]LGE2, can only be generated through free radical oxidation of arachidonic acid

phospholipids. Iso[4]LGE2 is a highly reactive γ-ketoaldehyde which modifies the ε-

amino groups of lysine residues forming protein-bound pyrroles lactams and

hydroxylactams,4 as well as protein-protein cross-links for which aminal5 and pyrrole-

pyrrole6-7 structures have been postulated, as well as DNA-protein cross-links.8

Incubation of salmon sperm DNA with LGE2 causes DNA-protein cross-linking in

cultured cells (10 mM MOPS buffer), suggesting that levuglandins can directly react with

81

DNA. Cyclopentenone PGs contain α,β-unsaturated carbonyl groups that readily adduct

various biomolecules such as glutathione (GSH) in vitro (Scheme 3.1).9

O OH

COOH

PGJ2

+

NHCH2OCH2CH2CHCOOHNH2

CHCONHCH2COOH

CH2SH

GSH transferase


CHCONHCH2COOH

CH2S

O OH

COOH

+ 2H ·


CHCONHCH2COOH

CH2S

HO OH

COOH

GSH

Scheme 3.1 Schematic representation of pyrrole cross-linking structure in previous study:

adducts of PGJ2 reaction with GSH in HCA-7 cells.9

LGE2 also reacts with nucleotides in vitro to form covalent adducts with a

dihydroxypyrrolidine structure (Scheme 3.2).10

82

O O

OH

HOO

N

N

O

NH2

dR

N

N

O

NH

dR

OHR'R

O

HemiaminalH2O

ProteinsDNA

N

N

O

N

dR

R'R

O

N

N

O

N

dR

OH

R'R

HO

2 H2O

N

N

O

N

dR

R'R

Pyrrole

+ O2

ProteinsN

N

O

N

R'R

lysine

O

Lactam

Dihydroxypyroolidine Schiff base


adducts of nucleosides and DNA formed by reaction with levuglandin.10

PGH2 reacts with arginine to yield covalent adducts (Scheme 3.3).11 These structures

would be appropriate candidates for isoLG/LG-protein cross-link.

83

O OH

COOH

LGE2

H

OH2N CHCCH2

OHO

CH2CH2NHCNH2

NH

Arginine

+

H2N CHCCH2

OHO

CH2CH2N

CN N LGE2LGE2

hydrolysis

COOH

N

HOOC

N

O

O

O

H2N CHCCH2

OHO

CH2CH2NH2

Ornithine

+


formation of bis(levuglandinyl) urea.11

IsoLGs initially form reversible Schiff base adducts12-13 with proteins which can

dehydrate to pyrroles. These adducts can be further oxidized to form stable lactams and

hydroxylactams. A possible cross-linking mechanism identifies the LG-protein Schiff

base adduct as the reactive electrophile that binds with a primary amino group in a

second molecule of protein resulting in aminal formation.7 IsoLG/LG cross-link forms

also include pyrrole further reaction with nucleophiles like unoxidized pyrroles (Scheme

3.4)4 or thiols (Scheme 3.5).14-15

N

R R

N

R R

N

R R

O2 NH

O2NH

NH


nucleophilic substitution of alkylpyrroles in the presence of oxygen.4

84

O

O

NH

O HN

OOR

O

H

NH2

NH

O HN

OOR1

O

H

N

N

NH

OR2

O

OHN

O

H

+

R= H, CH3

R1 = R2 = HR1 = H, R2 = CH3

(A)

O

O

HN

O

COOH

H CH2SH

HN

O

COOH

H CH2

NH2

HN

O

COOH

S

N

CH2HNH

COOH

O

(B)

Scheme 3.5 Schematic representation of pyrrole cross-linking structures in previous

studies. (A) Pyrrole autoxidation products in 2,5-hexanedione-treated dipeptide.14 (B)

Formation of thiol-pyrrole conjugates.15

The different ways in which isoLGs have been shown or postulated to modify

proteins is depicted in Scheme 3.6. IsoLGs modify proteins in a matter of seconds and

cross-link them within minutes, which is much faster than the rates at which HNE or

MDA form cross-links.6 Modification of the proteasome and other intracellular proteins

targeted by isoLGs can lead to impaired degradation.16-18 For example, isoLG-adducted

ovalbumin and Aβ1-40 aggregates significantly inhibit the proteasome activity of

neuroglial cultured cells and induce cell death, suggesting that isoLG/LGs may have

85

relevance to the pathogenesis of neurotoxicity19 and neurodegenerative diseases.17 Also

these adducts have been shown to hinder the activity of the 20S proteasome, a component

of cytosolic protein, degradation machinery in a dose dependent manner.17 Accumulation

of iso[4]LGE2-modified proteins has been observed in many diseases such as

artherosclerosis20 and rheumatoid arthiritis.21

Scheme 3.6 Schematic representation of formation of iso[4]levuglandin protein adducts

and cross-links.

OO

CH3

Protein

ON

CH3

Schiff base

Protein

HO-PC

N

H3C

Pyrrole

Bispyrrole Crosslink

N

H3C OH

NH

Protein-protein Aminal Crosslink

Protein

Protein

Protein

O2 ROO

N

H3C

Protein

OH

O

Hydroxylactam+

N

H3C

Protein

O

Lactam

CC5H11

OH

OO-PC

CO

O-PCC5H11

OH

CC5H11

OOH

OH

CC5H11

OOH

OH

CC5H11

OH

CC5H11

OH

OOH

OOH

N

N

COOH C5H11

OH

C5H11COOH

HOProtein

Protein

Lys NH2

Protein Lys NH2

C5H11

AA-PC

(CH2)3COO

Iso[4]LGE2

-H2O

O2

O2

H2O

OPO(CH2)2NMe3

RCOO

O

O

86

3.1.3 β-Amyloid and isolevuglandins

Alzheimer’s disease (AD) brain is under intense oxidative stress, manifested by

increased protein oxidation, lipid peroxidation, free radical formation, DNA/RNA

oxidation, nitrotyrosine levels, and advanced glycation end products.22-23 Cerebral

accumulation of soluble oligomers of (Aβ) 1-42 that are cytotoxic to neurons is central to

the pathogenesis of Alzheimer’s disease (AD).24-28 Addition of Aβ (1-42) to neurons

leads to increased protein carbonyls and decreased cell survival compared to controls.29-34

Protein oxidation is increased in AD brain in regions rich in Aβ (1-42). In parallel,

evidence has indicated long-term use of inhibitors of the cyclooxygenases is associated

with a decrease in the severity of Alzheimer’s disease. Meantime PGH2 accelerates the

formation of oligomers of Aβ1-42 in conjunction with lipid modification of the protein. It

provides a molecular basis for the hypothetical mechanism that links cyclooxygenase

activity to the formation of oligomers of Aβ.16 Aggregation of amyloid β (Aβ) 1-42

peptide forming neurotoxic oligomers may be a consequence of the LG-protein-protein

cross-linking that was observed upon treatment of Aβ 1-42 with LGE2.35

87


3.2.1 Reactions of model peptides with iso[4]LGE2.

Iso[4]LGE2 or iso[4]LGE2-FluoTag was incubated with N-acetylglycyllysine methyl

ester or β-amyloid (11-17) peptide “EVHHQKL” individually in NEM·OAc (pH 8.6) at

25°C for several days. Reactions were also conducted in both acidic (50 mM phosphate

buffer, pH 5.8) and neutral (50 mM phosphate buffer, pH 7.0) solutions. The results were

similar as in basic buffer. The reaction product profile exhibited no relationship with

buffer pH (vide infra). The resulting mixtures were dried on a speed vacuum, and re-

suspended in 10% acetonitrile/water with a trace of formic acid (0.1%) and analyzed by

mass spectrometry.

3.2.2 Mass spectrometric analysis of the iso[4]LGE2-peptide adducts.

N-Acetyl-glycyl-lysine methyl ester (N-acetyl-Gly-Lys-OMe) and β-amyloid(11-17)

peptide “EVHHQKL” were used as models for characterizing the modification of protein

lysyl residues resulting from adduction of iso[4]LGE2. A preliminary analysis of the

reaction product mixtures using MALDI-TOF mass spectrometry revealed the presence

of numerous products of peptide modification and indicated that both β-amyloid(11-17)

and N-acetylglycyllysine methyl ester showed similar products of adduction. Further

analysis was performed using triple quadrupole by ESI-MS and Q-TOF by ESI-MS/MS

to characterize the products.

(i) Iso[4]LGE2 modification of N-acetyl-gly-lys methyl ester.

MALDI-TOF MS analysis of the reaction product mixture from iso[4]LGE2 with a

large excess of N-acetylglycyllysine methyl ester showed major ions at m/z 576, 592,

88

608, 853, and 1149 corresponding to protonated pyrrole, lactam, hydroxylactam, aminal

cross-link and bispyrrole in addition to ions resulting from the loss of one or two

molecules of water from those adducts. Figure 3.1 shows a representative spectrum

revealing the presence of for various adducts in the reaction product mixture (see

appendix Table 3.1S for the molecular weights of various iso[4]LGE2-(N-acetyl-gly-lys-

OMe) adducts).

The reaction products for various reactant molar ratios were recorded (see appendix

Figure 3.1S (1) for N-acetyl-glycyl-lysine methyl ester: iso[4]LGE2 = 2000:1, (2) for

1000:1 and (3) for 200:1 in 1, 4, 7, 15, 21 and 50 day reactions). The initial concentration

of N-acetyl-glycyl-lysine methyl ester was constant while various ratios of iso[4]LGE2

were added. At various incubation times, identical aliquots of reaction mixture were

purified by Ziptip and then analyzed.

One-to-one adducts, i.e., pyrrole, lactam and hydroxylactam and their dehydration

products were generated very quickly (within one day reaction) in the reaction of

iso[4]LGE2 with N-acetyl-glycyl-lysine methyl ester (see appendix Figures 3.1S 1A).

Cross-link adducts included aminals, bispyrroles, trispyrroles and their dehydrated

forms. With higher iso[4]LGE2 ratio and longer incubation times, a higher percentage

(comparison of cross-link peak intensity with one-to-one adducts) of bispyrrole adducts

was generated. Specifically, bispyrrole peak intensity was higher when the molar ratio of

N-acetyl-glycyl-lysine methyl ester to iso[4]LGE2 was 200:1 than 2000:1 (compare

appendix Figures 3.1S 3A-F). This suggests that the dipeptide interferes with pyrrole-

pyrrole cross-link formation. This interpretation is also consistent with the results of later

experiments (see Chapter 4, section 4.2.4) testing the effect of dipeptide on the oxygen-

89

promoted dimerization and adduct formation reactions of pure pyrrole. Bispyrrole peak

intensity was also higher when N-acetyl-glycyl-lysine methyl ester reaction time with

iso[4]LGE2 was 15 days versus 1 day.

Derivatives of the polypyrroles, i.e., bispyrrole and trispyrrole showed a sequence of

ions corresponding to further addition of oxygen atoms (+16). Each polypyrrole parent

ion is accompanied by a series of ions corresponding to the addition of one or more atoms

of oxygen to the polypyrrole. The details are discussed below.

Figure 3.1 MALDI-TOF MS analysis of the reaction mixture from N-acetylglycyllysine

methyl ester and iso[4]LGE2 (molar ratio = 200:1) (50 mM NEM∙OAc aqueous solution

with 4% methanol, v/v, pH 8.6) at 25 °C for 4 days.

90

The product mixture from reaction of iso[4]LGE2 with N-acetyl-gly-lys-OMe was

separated by reverse phase HPLC using an acetonitrile/water gradient and monitored by

LC-ESI extracted ion channels (Figure 3.2). The major products corresponding to lactam

(trace 3.2C), aminal cross-link (trace 3.2B) and bispyrrole (trace 3.2D) eluted in order of

decreasing polarity at about 12, 30, and 36 minutes.

A

TIC

BispyrroleD

B

C

AminalCrosslink

Lactam-H2O

m/z 854

m/z 1150

m/z 574

6.8e8

1.3e6

5.8e6

3.7e6

Figure 3.2: LC-ESI for the product mixture from the reaction of N-acetylglycyllysine

methyl ester with iso[4]LGE2 (molar ratio = 200:1 at 25 °C for 4 days) with selected ion

recording for aminal cross-link, lactam-H2O and bispyrrole. A) TIC, B) aminal cross-link

m/z 854.1 (+ H+), C) lactam-H2O m/z 574.7 (+ H+), D) bispyrrole m/z 1150.5 (+ H+).

MALDI-TOF MS analysis of the fractions eluting at about 12, 30 and 36 minutes

confirmed the presence of the lactam, aminal and bispyrrole cross-links. Thus, MALDI-

TOF MS analysis of the 30 minute elution showed major ions at m/z 853.5, 835.5 and

817.5 corresponding to protonated aminal cross-link in addition to ions resulting from the

loss of one and two molecules of water. MALDI-TOF MS analysis of the 36 minute

91

elution showed major ions at m/z 1149.7, 1131.7 and 1113.7 corresponding to protonated

bispyrrole in addition to ions resulting from the loss of one and two molecules of water.

The MALDI-TOF mass spectra of these principle products are shown in Figure 3.3. A

series of ions are present in the mass spectrum of the isoLG-N-acetylglylys-OMe

bispyrrole cross-link chromatographic fraction corresponding to the addition of one or

more atoms of oxygen to the bispyrrole, and the relative amount of these products of

further oxidation is greater after incubation for 15 days compared to 4 days (Figure 3.3

panels A and B, respectively). The peak intensity of further oxidized bispyrrole (products

with addition of oxygen atoms) was higher compared with bispyrrole and its dehydrated

forms after 15 days modification than after 4 days. The pattern of further addition of

oxygen atoms (+16) was also observed for the trispyrrole products. Thus, pyrrole

oligomerizes to form polypyrroles, and all of these products undergo further oxidation.

Possible structures for products of oxidized bispyrrole (+16, +32, +48, +64) were shown

in Scheme 3.7.

N

N

OR2

R1

Peptide

Peptide

R1

R2

+16

N

O

N

OR2

R1

Peptide

Peptide

R1

R2

+32

N

O

N

OR2

R1

R1

R2

+48 +64

O

N

O

N

OR2

R1

R1

R2

O

O

Peptide

Peptide

Peptide

Peptide

Scheme 3.7 Possible structures for products of oxidized bispyrrole (+16, +32, +48, +64).

Previously, singlet oxygen, a major intermediate in the photodynamic effect,

generated by the combined action of light, a photosensitizing dye and oxygen, was

believed to insert oxygen atoms into biological substrates.36 For example, singlet oxygen

92

reacts with double bonds giving epoxides.37 The pyridinium bisretinoid A2E, a major

hydrophobic fluorophore of retinal pigment epithelium lipofuscin undergoes such

addition of multiple atoms of oxygen. Thus, a series of oxidized derivatives is generated

upon 10 min of blue light irradiation, each of which represents the addition of an oxygen

atom at a carbon-carbon double bond. A2E self-generates singlet oxygen with the singlet

oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bonds

(Figure 3.4). Epoxidation occurs at all nine double bonds in the hydrocarbon side chains

without modifying the pyridinium ring.38-39

A B

C D

+O

+O+O

+O

+O-18

-18

-18

-18

+O+O

+O

+O

+O

+O

-18

-18

-18-18

-18

Figure 3.3 MALDI-TOF MS spectra: panel A (4 days reaction) and B (15 days reaction)

for 36 min HPLC fractions, panel C (15 days reaction) for 30 min HPLC fraction, panel

93

D (15 days reaction) for 41 min HPLC fraction of products from the reaction of N-

acetylglycyllysine methyl ester with iso[4]LGE2 (molar ratio = 200:1 at 25 °C for 4 and

15 days).

A2E 1

Figure 3.4 Top: Structures of A2E (1), the bisoxirane (2) and nonaoxirane (3). Bottom:

ESI mass spectrum of A2E after illumination with blue light (430 nm; 10 min) to yield a

mixture of various oxygenated derivatives resulting in a series of ions that differ in m/z

by 16 owing to the addition of oxygen atoms to A2E. The addition of oxygen,

94

presumably to form epoxides, occurred at carbon-carbon double bonds of A2E, measured

by Micromass ESI-Q-TOF.38-39

To study their fragmentation patterns, tandem MS/MS analyses of pyrrole, lactam,

hydroxylactam and aminal cross-link ions were carried by Q-TOF mass spectrometry.

ESI-MS/MS analysis of Zip-Tip purified reaction mixture from N-acetylglycyllysine

methyl ester and iso[4]LGE2 (molar ratio = 200:1 at 25 °C for 4 days) provided additional

support for the structure postulated for the aminal cross-link. Previously, it was

postulated that an initial iso[4]LGE2 Schiff base adduct with the lysine residue of N-

acetyl-gly-lys-OMe is an electrophilic intermediate that undergoes aminal cross-linking

with another nucleophilic side chain residue of a second molecule of N-

acetylglycyllysine methyl ester.5 The MS/MS spectrum of the parent ion from the

putative aminal cross-link (m/z 853.5) shows peaks at m/z 853.5 and 835.5 corresponding

to the [M+H]+ ion from iso[4]LGE2-(N-acetyl-gly-lys-OMe) aminal cross-link and its

dehydrated form (Figure 3.5B). The MS/MS spectrum of m/z 835.5 exhibits two

diagnostic daughter ions at m/z 558.4 and 436.3. The ion at m/z 558.4, which is isobaric

with protonated dehydrated pyrrole, corresponds to loss of one molecule of N-acetyl-gly-

lys-OMe (-259) and one molecule of H2O (-18). The ion at m/z 436.3 corresponds to loss

of non-3-en-1-yne (C9H14) (-122). The proposed structure of each daughter ion is shown

in Figure 3.5 and Scheme 3.8. See appendix Figure 3.2S and Scheme 3.1S for spectra and

fragmentation structures of the pyrrole, lactam and hydroxylactam one-to-one adducts.

95

m/z

TOF MSMS 835.55ES+

TOF MSMS 853.58ES+

A

B

Relative Abundance

Relative Abundance

m/z

N CC5H11

OHO

HN

ONH

O COOCH3

OH

OH

NH

HN

NH

O

O COOCH3

H+N C

C5H11

OHO

HN

ONH

O COOCH3OH

NH

HN

NH

O

O COOCH3

H+

N CC5H11

OHO

HN

ONH

O COOCH3H+

N C OHO

HN

ONH

O COOCH3

H+

Figure 3.5 ESI-MSMS of molecular ions at A) m/z 835 (+ H+), and B) m/z 853 (+ H+)

corresponding to an iso[4]LGE2-(N-acetyl-gly-lys-OMe) aminal cross-link and its

dehydrated form.

N CC5H11

OHO

HN

ONH

O COOCH3

OH

OH

NH

HN

NH

O

O COOCH3835.5 (M - H2O + H+)

558.4 (835.5 - NGL - H2O)

558.4 (835.5 - NGL - H2O)

436.3 (558.4 - C9H14)

Scheme 3.8 Possible fragmentation of an iso[4]LGE2-(N-acetyl-gly-lys-OMe) aminal

cross-link at m/z 853.5 (M + H+).

96

(ii) Modification of N-acetyl-gly-lys methyl ester with fluorescently labeled iso[4]LGE2.

MALDI-TOF MS analysis of the reaction product mixture from the reaction of

fluorescently labeled iso[4]LGE2 (iso[4]LGE2-FluoTag) with N-acetylglycyllysine

methyl ester showed major ions at m/z 843, 859, 875 and 1120 corresponding to

protonated pyrrole, lactam, hydroxylactam and aminal cross-link, respectively, in

addition to ions resulting from the loss of one or two molecules of water from those

adducts. Figure 3.6 shows a representative spectrum showing the presence of various

adducts (see appendix Table 3.1S for molecular weight of adducts of N-acetyl-gly-lys-

OMe with iso[4]LGE2-FluoTag).

The reaction products were recorded (see appendix Figure 3.3S (1) for two different

reactant molar ratios of N-acetyl-glycyl-lysine methyl ester: iso[4]LGE2-FluoTag =

2000:1 and (2) for 200:1 measured after incubation for 1, 4, 15, 21 and 50 days). The

initial concentration of N-acetyl-glycyl-lysine methyl ester was constant while two

different ratios of iso[4]LGE2-FluoTag were added. At various incubation times, identical

aliquots of reaction mixture were purified by Ziptip and analyzed.

The 1:1 adducts, i.e., pyrrole, lactam and hydroxylactam and their dehydrated forms

were generated very quickly (within one day reaction) in the reaction of iso[4]LGE2-

FluoTag with N-acetyl-glycyl-lysine methyl ester (see appendix Figure 3.3S 1A). For the

aminal cross-link and its dehydrated forms, with longer incubation times, a higher

percentage of aminal cross-link adducts versus 1:1 adducts was generated. Specifically,

the aminal cross-link peak intensity was higher when N-acetyl-glycyl-lysine methyl ester

reaction time with iso[4]LGE2-FluoTag was 15 days than just 1 day (see appendix

Figures 3.3S 2A-E).

97

MALDI-TOF MS analysis was performed on 100 fmol of Zip-Tip purified reaction

mixture. Bispyrrole couldn’t be detected. Higher loading amount (200-500 fmol) was

also tested, but no bispyrrole was detected either. Thus, whereas bispyrrole is a minor but

significant product from the reaction of iso[4]LGE2 with N-acetylglysyllysine methyl

ester, it is not even a detectable product from the reaction of iso[4]LGE2-FluoTag with

this peptide. The difference between these adducts is isoLG ester versus carboxylate. One

possibility is that the carboxylate interacts with a putative cation radical intermediate and

alters its proclivity for combination with O2 versus a second molecule of pyrrole.

Figure 3.6 MALDI-TOF MS analysis of the product mixture from the reaction of N-

acetylglysyllysine methyl ester with iso[4]LGE2-FluoTag (molar ratio = 200:1) (50 mM

NEM∙OAc aqueous solution with 6% methanol, v/v, pH 8.6) at 25 °C for 15 days.

98

An investigation of the fragmentation patterns from tandem MS/MS analysis of

aminal cross-link ions by Q-TOF mass spectrometry provided additional support for the

structure postulated for this type of cross-link. The initial Schiff base of iso[4]LGE2-

FluoTag with the lysine residue of N-acetylglycyllysine methyl ester is an electrophilic

intermediate that undergoes aminal cross-linking with another nucleophilic side chain

residue of a second molecule of N-acetyl-gly-lys-OMe. The MS/MS spectrum of the

parent ion from the putative aminal cross-link (m/z 1120.6) shows peaks at m/z 1120.6

and 1102.5 corresponding to the [M+H]+ ion from a iso[4]LGE2-FluoTag-(N-acetyl-gly-

lys-OMe) aminal cross-link and its dehydrated form (Figure 3.7B). The MS/MS spectrum

of m/z 1102.5 exhibits two diagnostic daughter ions at m/z 825.5 and 703.4. The ion at

m/z 825.5, which is isobaric with protonated dehydrated pyrrole, corresponds to loss of

one molecule of N-acetyl-gly-lys-OMe (-259) and one molecule of H2O (-18). The ion at

m/z 703.4 corresponds to loss of non-3-en-1-yne (C9H14) (-122). The proposed structure

of each daughter ion is shown in Figure 3.7 and Scheme 3.9. See appendix Figure 3.4S

and Scheme 3.2S for spectra and fragmentation structures of one-to-one adducts: pyrrole,

lactam and hydroxylactam.

99

Figure 3.7 ESI-MSMS of molecular ions at A) m/z 1102.5 and B) m/z 1120.6

corresponding to a iso[4]LGE2-FluoTag-(N-acetyl-gly-lys-OMe) aminal cross-link

adduct and its dehydrated form.

N CC5H11

NHCH2CH2NHO

HN

ONH

O COOCH3

OH

OH

NH

HN

NH

O

O COOCH31102.6 (M - H2O + H+)

825.5 (1102.6 - NGL - H2O)

825.5 (1102.6 - NGL - H2O)

703.4 (825.5 - C9H14)

NO

N

SO2N

Scheme 3.9 Possible fragmentation of iso[4]LGE2-FluoTag aminal cross-link with N-

acetyl-gly-lys-OMe at m/z 1120.6 (M + H+).

TOF MSMS 1102.5ES+

TOF MSMS 1120.6ES+

A

B

m/z

100

(iii) Modification of the β-amyloid(11-17) peptide “EVHHQKL” by iso[4]LGE2.

MALDI-TOF MS analysis of the reaction product mixture from iso[4]LGE2 with β-

amyloid(11-17) peptide “EVHHQKL” showed major ions at m/z 1206, 1222, 1238, 2114

and 2410 corresponding to protonated pyrrole, lactam, hydroxylactam, aminal cross-link

and bispyrrole, respectively, in addition to ions resulting from the loss of one or two

molecules of water at m/z 1188, 1204, 1220, 1202, 2096, 2078, 2392, 2374. Figure 3.8

shows a representative spectrum that shows ions corresponding to various adducts (see

appendix Table 3.1S for the molecular weights of various iso[4]LGE2-β-amyloid

(EVHHQKL) peptide adducts).

Figure 3.8 MALDI-TOF MS analysis of the product mixture from the reaction of β-

amyloid(11-17) peptide “EVHHQKL” with iso[4]LGE2 (molar ratio = 200:1) (50 mM

NEM∙OAc aqueous solution with 4% methanol, v/v, pH 8.6) 25 °C for 7 days.

101

Reaction products were recorded for two reactant molar ratios (see appendix Figure

3.5S (1) for β-amyloid (EVHHQKL) peptide: iso[4]LGE2 = 2000:1 and (2) 200:1

measured after incubation for 1, 4, 7, 15, 21 and 50 days). The initial concentration of β-

amyloid (EVHHQKL) peptide was the same for both reactions while various ratios of

iso[4]LGE2 were added. At various incubation times, identical aliquots of the reaction

mixture were purified by Ziptip and then analyzed.


were generated very quickly (within one day) in the reaction of iso[4]LGE2 with β-

amyloid (EVHHQKL) peptide (see appendix Figure 3.5S 1A). Cross-link adducts

included aminals, bispyrroles, trispyrroles and their dehydrated forms. With higher

iso[4]LGE2 ratio and longer incubation times, a higher percentage (comparison of cross-

link peak intensity with one-to-one adducts) of bispyrrole adducts generated. Specificity,

bispyrrole peak intensity was higher when the molar ratio of β-amyloid (EVHHQKL)

peptide to iso[4]LGE2 was 200:1 than 2000:1 (see appendix Figure 3.5S 2A-F).

Bispyrrole peak intensity was also higher (5 % more) when β-amyloid (EVHHQKL)

peptide reaction time with iso[4]LGE2 was 15 days than just 1 day. A similar trend was

observed with the dipeptide. It agrees with the conclusion the dipeptide or “EVHHQKL”

peptide interferes with bispyrrole formation. Presumably for the same reason, i.e. that

these peptides compete with a second molecule of pyrrole for a putative cation radical

intermediate.

More cross-link product versus bispyrrole formed during dipeptide reaction with

iso[4]LGE2. In contrast, more bispyrrole versus aminal cross-link product formed during

the reaction of “EVHHQKL” with iso[4]LGE2. It is tempting to speculate that the

102

“EVHHQKL” peptide-pyrrole modification forms aggregates that favor the dimerization

to produce bispyrrole since β-amyloid tends to spontaneously oligomerize. Future studies

might seek evidence for noncovalent aggregation of “EVHHQKL” peptide-pyrroles

under oxygen free conditions where oxidative transformations are precouded.

Polypyrroles, i.e., bispyrrole and trispyrrole showed a sequence of ions corresponding

to the further addition of oxygen atoms (+16). A series of peaks corresponding ions for

polypyrroles that had undergone the addition of one or more atoms of were observed. The

details will be discussed below.

The product mixture from reaction of iso[4]LGE2 with β-amyloid(11-17) peptide

“EVHHQKL” was separated by reverse phase HPLC using an acetonitrile/water gradient

and monitored by LC-ESI extracted ion channels (Figure 3.9). The major products

corresponding to aminal cross-link (trace 3.2C) and bispyrrole (trace 3.2B) eluted in

order of decreasing polarity at about 22 and 32 minutes.

Bispyrrole

Aminal crosslink

A

B

C

TIC

m/z 804 (+3)

m/z 1058(+2)

7e6

5.2e8

3e6

Figure 3.9 LC-ESI for the reaction mixture from β-amyloid(11-17) peptide and

iso[4]LGE2 (molar ratio = 200:1 at 25 °C for 7 days) with selected ion recording for

103

bispyrrole and aminal cross-links. A) TIC, B) bispyrrole m/z 804.6 (+3), m/z 603.7 (+4),

C) aminal cross-link m/z 1058.2 (+2).

MALDI-TOF MS analysis of the fractions eluted at 22 and 32 minutes confirmed the

presence of the aminal and bispyrrole cross-links. MALDI-TOF MS analysis of the

aminal fraction showed major ions at m/z 2114.2, 2096.2 and 2078.2 corresponding to

protonated aminal cross-link in addition to ions resulting from the loss of one and two

molecules of water. MALDI-TOF MS analysis of the bispyrrole fraction showed major

ions at m/z 2410.3, 2392.3 and 2374.3 corresponding to protonated bispyrrole in addition

to ions resulting from the loss of one and two molecules of water. The mass spectra of the

principle products are shown in Figure 3.10. A series of peaks are present in the mass

spectrum of the isoLG-β-amyloid(11-17) peptide chromatographic fraction corresponding

to the addition of one or more atoms of oxygen to the bispyrrole, and the relative amount

of these products of further oxidation as indicated by the relative peak intensity of further

oxidized bispyrrole (products with addition of oxygen atoms) compared with bispyrrole

and its dehydrated forms after 15 days modification than after 7 days (Figure 3.10 panels

A and B, respectively).

A series of products of further addition of oxygen (multiple +16 m/z increments)

pattern was also observed for the trispyrrole adduct. Thus, pyrrole oligomerizes form

polypyrroles, and all of these products undergo further oxidation. Similar behavior was

observed for the iso[4]LGE2-(N-acetyl-gly-lys-OMe) bispyrrole cross-link and discussed

in section “(i) Iso[4]LGE2 modification of N-acetyl-gly-lys methyl ester”.

104

A B

C

-18

-18

-18

-18

-18

-18

D

Figure 3.10 MALDI-TOF MS spectra: panel A (7 days reaction) and B (15 days

reaction) for 32 min HPLC fractions, panel C and D (7 days reaction) for 22 min, 15 min

HPLC fraction of products from reaction of the β-amyloid(11-17) peptide “EVHHQKL”

with iso[4]LGE2 reaction (molar ratio = 200:1 at 25 °C).

(iv) Modification of β-amyloid(11-17) peptide “EVHHQKL” with fluorescently labeled

iso[4]LGE2.

MALDI-TOF MS analysis of the reaction product mixture from iso[4]LGE2-FluoTag

with the β-amyloid (EVHHQKL) peptide showed major ions at m/z 1473, 1489 and 1505

corresponding to protonated pyrrole, lactam and hydroxylactam in addition to ions

105

resulting from the loss of one or two molecules of water from those adducts. Figure 3.11

is a representative spectrum that shows ions corresponding to various adducts (see

appendix Table 3.1S for molecular weight of adducts of β-amyloid (EVHHQKL) peptide

with iso[4]LGE2-FluoTag).

Reaction products were recorded for two reactant molar ratios (see appendix Figure

3.6S (1) for β-amyloid (EVHHQKL) peptide: iso[4]LGE2-FluoTag = 2000:1 and (2)

200:1 measured after incubation for 1, 4, 15, 21 and 50 days. The initial concentration of

β-amyloid (EVHHQKL) peptide was the same for both reactions while various ratios of

iso[4]LGE2-FluoTag were added. At various incubation times, identical aliquots of

reaction mixture were purified by Ziptip and analyzed.


were generated very quickly (within one day) in the reaction of iso[4]LGE2-FluoTag with

β-amyloid (EVHHQKL) peptide (appendix Figure 3.6S 1A). MALDI-TOF analysis was

performed on 100 fmol of Zip-Tip purified reaction mixture. Compared with 1:1 adducts,

aminal cross-link and bispyrrole were barely be detected.

106

Figure 3.11 MALDI-TOF MS analysis of the product mixture from the reaction β-

amyloid (EVHHQKL) peptide with iso[4]LGE2-FluoTag (molar ratio = 200:1) (50 mM

NEM∙OAc aqueous solution with 6% methanol, v/v, pH 8.6) at 25 °C for 15 days.

107

3.3 Conclusions


aggregation. N-Acetyl-glycyl-lysine methyl ester (N-acetyl-Gly-Lys-OMe) and β-

amyloid(11-17) peptide “EVHHQKL” were used as models for characterizing the cross-

linking of protein lysyl residues resulting from adduction of iso[4]LGE2. Aminal,

bispyrrole and trispyrrole cross-links of two model peptides, were identified and fully

characterized by mass spectrometry, and a large variety of derivatives of the bispyrrole

cross-link containing one or more additional atoms of oxygen were discovered. More

aminal cross-link product versus bispyrrole is formed during dipeptide reaction with

iso[4]LGE2, and bispyrrole is almost undetectable in the reaction of a fluorescently

labeled iso[4]LGE2 with dipeptide. In contrast, more bispyrrole versus aminal cross-link

product formed during the reaction of “EVHHQKL” with iso[4]LGE2. It is tempting to

speculate that the “EVHHQKL” peptide-pyrrole modification forms aggregates that favor

the dimerization to produce bispyrrole since β-amyloid tends to spontaneously

oligomerize. Future studies might seek evidence for noncovalent aggregation of

“EVHHQKL” peptide-pyrroles under oxygen free conditions where oxidative

transformations are precouded.

With higher iso[4]LGE2 ratio:peptide, a higher percentage of bispyrrole versus 1:1

adducts was generated from both N-acetyl-glycyl-lysine methyl ester and β-amyloid

(EVHHQKL) peptide. This agrees with the conclusion that these peptides interfere with

bispyrrole formation. Presumably for the same reason, i.e. that these peptides compete

with a second molecule of pyrrole for a cation radical intermediate that will be discussed

in the following chapter.

108



following commercially available materials were used as received: N-acetylglycyllysine

methyl ester was from Bachem (Torrance, CA), β-Amyloid (11-17) peptide

“EVHHQKL” was from Peptide 2.0 Inc (Chantilly, VA), N-ethylmorpholine was from

Aldrich (Milwaukee, WI), and all other chemicals were obtained from Fisher Scientific

Co (Chicago, IL). Iso[4]LGE2 was prepared by Dr. Jim Laird and iso[4]LGE2-FluoTag

was prepared as described in Chapter 2. Chromatography was performed with ACS grade

solvents.

3.4.2 Peptide modification with iso[4]LGE2 or iso[4]LGE2-FluoTag. Reactant stocks

were prepared as follows: 25 mM N-acetylglycyllysine methyl ester and β-amyloid (11-

17) peptide “EVHHQKL” in NEM∙OAc (50 mM, pH 8.6), 1 mg/ml iso[4]LGE2 (without

or with fluorescence tag) in MeOH.

4.4 µl of iso[4]LGE2 (12.5 nmol, 4.4 µg) or 7.7 µl of iso[4]LGE2-FluoTag (12.5

nmol, 7.7 µg) was added to N-acetylglycyllysine methyl ester (2.5 µmol, 647.5 µg) in 50

mM pH 8.6 NEM∙OAc (100 µl) (molar ratio of iso4: dipeptide = 1:200) and reacted

under air on shaker (IKA MTS 2/4 digital microtiter shaker, 500 rpm) at 25 °C. At

various times (1, 4, 7, 15, 21 and 50 days), an aliquot (~1.2 nmol, 10 µl) of reaction

mixture was quickly dried using a high-speed vacuum evaporator and then stored at -20

°C until analysis. The aliquot was analyzed with a MALDI-TOF, triple quadrupole or Q-

TOF mass spectrometer. Two different molar ratios (iso4: peptide = 1:2000 and 1:200)

were studied. β-amyloid (11-17) peptide “EVHHQKL” was treated the same way.

109

3.4.3 Analysis of adducts using MALDI-TOF. Initial diagnostic analysis of the samples

was performed by MALDI-TOF mass spectrometry on an AB Sciex4800 Plus MALDI

TOF/TOF™ Analyzer equipped with a UV laser (355 nm) and reflector mode with a

matrix of α-cyano-4-hydroxycinnamic acid (CHCA, 10 mg/ml in acetonitrile/water/3%

formic acid, 5:4:1, v/v/v). A peptide mass standards kit for calibration of AB Sciex

MALDI-TOFTM instruments was used to calibrate the plate. Each of the reaction

aliquots was resuspended in 10% aqueous acetonitrile solution and vortexed to mix well

before C18 Ziptip purification (Millipore, ZipTip with 0.6 µL C18 resin). To wet the C18

Ziptip pipettor plunger was depressed to a dead stop using the maximum volume setting

of 20 µl. Acetonitrile (wetting solution) was aspirated into tip then dispensed to waste

and the process repeated twice. The tip was equilibrated for binding by washing three

times with the 0.1% formic acid/water (equilibration solution). Samples were bound to

ZipTip by fully depressing the pipettor plunger to a dead stop then aspirating and

dispense the sample 3 to 7 cycles. The tip was then washed with at least 3 cycles of 0.1%

formic acid/water (wash solution) dispensing to waste using. The samples were eluted

with 5 µl of 80% acetonitrile/0.1% formic acid (elution solution) into a clean vial to give

a final concentration of 100 fmol/µl by aspirating and dispensing eluant through the

ZipTip at least 3 times without introducing air. Two microliters of the sample was then

mixed with 1 µl of matrix, and then 1.5 µl were applied to the plate spot and allowed to

dry. The data was processed with Data Explorer 4.0 (AB Sciex) software.

110

Table 3.1: Optimized parameters for MALDI-TOF mass spectrometer.

Acquisition

Method

Parameters Data Parameters Data Shots/sub-spectrums 50 Bin size (ns) 0.5 Total shots/spectrum 1000 Input bandwidth (MHZ) 500 Laser intensity 3000 Detector voltage multiplier 0.92 Vertical scale (v) 0.5 Final detector voltage (KV) 2.015

Processing

Method

Calibration Parameters Data Peak Detection Parameters Data Min S/N 20 Min S/N 10 Mass tolerance (m/z) 2 Local noise window width (m/z) 250 Min peaks to match 4 Min peak width (bins) 2.9 Max error (ppm) 100 Mass resolution 22000

3.4.4 LC-MS (triple quadrupole) analysis. Iso[4]LGE2-modified β-amyloid(11-17)

peptide was separated by HPLC with a 300 µm x 250 mm i.d. C18 5 µm column

(DIONEX AcclaimTM). Chromatography was carried out with linear elution gradient

(elute A, 0.1% TFA/H2O; elute B, 0.1% TFA/ACN) at a flow rate of 20 µl/min. The

samples were loaded onto the column with 2% B for 10 min. The gradient was as

follows: 2-12% B over 5 min, to 60 % over 48 min, to 98% B over 2 min, and hold for 10

min. Iso[4]LGE2 modified acetyl-gly-lys-O-methyl ester was seperated by HPLC with a

2.1 x 100 mm i.d. C18 1.8 µm column (Waters Acquity UPLC HSS). Chromatography

was carried out with linear elution gradient (elute A, 0.1% FA/H2O; elute B, 0.1%

FA/ACN) at a flow rate of 70 µl/min. Sample was loaded onto the column with 2% B for

10 min. The gradient was as follows: 2-10% B over 2 min, to 98 % within 25 min, and

hold for 18 min. The effluent was monitored by ESI-MS/MS with a API-3000 triple

quadrapole electrospray mass spectrometer (Applied Biosystems Inc.) to determine the

retention time of different oxidized adducts. The instrument was operated in the positive

mode and high-pressure nitrogen was used as source gas and scanning from m/z 220-

2000 using the parameters listed in table 3.2.

111

Table 3.2: Optimized parameters for triple quadrupole mass spectrometer.

Parameters Data Declustering Potential (DP) 30 Focus Potential (FP) 250 Entrance Potential (EP) 10 Nebulizer Gas (NEB) 10 Curtain Gas (CUR) 8 Ion Spray Voltage (IS) 4000 Temperature (TEM) 200

3.4.5 LC- MS/MS (Q-TOF) analysis. The samples were analyzed by a quadrupole-time

of flight (Q-TOF-2) mass spectrometer (Micromass) equipped with MassLynxTM

acquisition and processing software. 2 pmol iso[4]LGE2 or iso[4]LGE2-FluoTag modified

N-acetylglycyllysine methyl ester (molar ratio of N-acetylglycyllysine methyl ester:

iso[4]LGE2 = 200:1, 25 °C for 4 days), prepared as described in Section 3.4.2, was

analyzed by Q-TOF. For sample preparation, 62.5 pmol of reaction mixture from the

reaction of iso[4]LGE2 with N-acetylglycyllysine methyl ester was resuspended in 250 µl

10% acetonitrile. An aliquot (8 µl, 2 pmol) of this solution was purified with a Zip-Tip as

described in section 3.4.3 and eluted in 10 µl of 80% acetonitrile/0.1% formic acid. This

purified eluate was quickly dried using a high-speed vacuum evaporator and then

dissolved in 15 µl of 5% acetonitrile/2% formic acid and injected into the Q-TOF. The

samples were infused using a 100 µl Hamilton syringe onto a capillary column

(PicoFritTM 0.075 x 70 mm, 15 µ tip ID; New Objective Inc., Woburn, MA) at 0.5 µl/min

using a syringe pump (Harvard apparatus, Pump 11) and ionized immediately using an

electrosprayer designed in-house. The mass spectrometer was operated in standard MS

and MS/MS switching mode. The MS data was collected for the mass range of 50-1500

m/z and MS/MS data was analysed with Micromass software (MassLynx) Version 3.5.

112

The machine was calibrated using a solution of 2 fmol of [Glu1]-Fibrinopeptide B

(Sigma) in 50% aqueous acetonitrile (+0.1% formic acid) infused using glass

micropipette with 5 µ tip. The intensity of the peak from the MS/MS spectrum at 785.4

m/z was used as a reference for calibration. The final mass measurement accuracy of ≤ 10

ppm was achieved before the use of the machine.

113

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32. Yatin, S. M.; Yatin, M.; Varadarajan, S.; Ain, K. B.; Butterfield, D. A. (2001)

Role of spermine in amyloid beta-peptide-associated free radical-induced neurotoxicity. J

Neurosci Res 63 (5), 395-401.

33. Varadarajan, S.; Kanski, J.; Aksenova, M.; Lauderback, C.; Butterfield, D. A.

(2001) Different mechanisms of oxidative stress and neurotoxicity for Alzheimer's A

beta(1--42) and A beta(25--35). J Am Chem Soc 123 (24), 5625-5631.

34. Yatin, S. M.; Varadarajan, S.; Butterfield, D. A. (2000) Vitamin E Prevents

Alzheimer's Amyloid beta-Peptide (1-42)-Induced Neuronal Protein Oxidation and

Reactive Oxygen Species Production. J Alzheimers Dis 2 (2), 123-131.

35. Boutaud, O.; Montine, T. J.; Chang, L.; Klein, W. L.; Oates, J. A. (2006) PGH2-

derived levuglandin adducts increase the neurotoxicity of amyloid beta1-42. J

Neurochem 96 (4), 917-923.

118

36. Straight, R. C.; Spikes, J. D., Singlet Oxygen. CRC: Boca Raton, FL, 1985; Vol.

4, 91.

37. Schulte-Elte, K. H.; Gadola, M.; Ohloff, G. (1973) Oxydative Umsetzungen in der

Damascon-Reihe. 1ΔgO2- und SeO2-Oxydation von β-Damascenon. Helv Chim Acta 56

(6), 2028-2035.

38. Ben-Shabat, S.; Itagaki, Y.; Jockusch, S.; Sparrow, J. R.; Turro, N. J.; Nakanishi,

K. (2002) Formation of a nonaoxirane from A2E, a lipofuscin fluorophore related to

macular degeneration, and evidence of singlet oxygen involvement. Angew Chem Int Ed

Engl 41 (5), 814-817.

39. Sparrow, J. R.; Vollmer-Snarr, H. R.; Zhou, J.; Jang, Y. P.; Jockusch, S.; Itagaki,

Y.; Nakanishi, K. (2003) A2E-epoxides damage DNA in retinal pigment epithelial cells.

Vitamin E and other antioxidants inhibit A2E-epoxide formation. J Biol Chem 278 (20),

18207-18213.

119

CHAPTER 4

GENERATION OF ISOLG-PYRROLE BY THE REACTION OF

ISOLG WITH N-ACETYL-GLY-LYS-OMe IN THE ABSENCE OF O2

AND THE OXIDATION OF ISOLG-PYRROLE TO FORM

PYRROLE-PYRROLE CROSS-LINKS AND AN ADDUCT WITH N-

ACETYLCYSTEINE

4.1 Background

4.1.1 Purpose

Presumably, pyrrole-pyrrole cross-links are produced by oxidative coupling of

LG/isoLG-pyrroles. To enable examination of the mechanism of isoLG-pyrrole-pyrrole

cross-linking, it is desirable to synthesize pure isoLG-pyrroles. In this chapter, the

preparation of pure isoLG-pyrroles and a study of their chemistry will be described.

Evidence is presented that oxidation and cross-linking of isoLG-derived pyrroles is

autocatalytic the initiation of which can be catalyzed by tetramethylpyrrolidine-N-oxide,

a stable free radical.

4.1.2 LGs and isoLGs modification with proteins

Previously, we discovered that the prostaglandin endoperoxide intermediate of the

cyclooxygenase pathway, and its stereo and structural isomers (isoprostane

endoperoxides), which are generated through free radical-induced oxidation of

arachidonyl phospholipids, undergo rearrangements that produce γ-ketoaldehydes,

levuglandins (LGs) and isoLGs, respectively. Adduction of LGs/isoLGs to proteins

initially produces pyrroles through hemiacetals within seconds. The pyrroles are readily

oxidized to lactams and hydroxylactams that are the major lysyl derivatives present in

proteins in vivo. LG/isoLG adduction also generates protein-protein cross-links within

minutes.1 Pyrrole-pyrrole cross-link and aminal cross-link structures shown in Scheme

4.1 have been postulated, but no direct molecular level evidence for the cross-link

structure(s) has been reported.

121

Scheme 4.1 LG/isoLG modification of proteins

122


4.2.1 Preparation of iso[4]LGE2-pyrrole under anerobic conditions.

As reported in chapter 3, the formation of pyrrole-pyrrole cross-links was detected

upon adduction of isoLG with N-acetyl-gly-lys methyl ester. Pyrrole-pyrrole dimer

formation is an oxidative process. Therefore, cross-linking is not expected to occur under

anerobic conditions. To test this hypothesis, the treatment of acetyl-gly-lys methyl ester

was conducted in an oxygen-free aqueous solution containing sodium dithionite, an

oxygen scavenger. As predicted, cross-linking was completely prevented. However,

immediately after removing Na2S2O4 (as describe below), the iso[4]LGE2-pyrrole

(Scheme 4.2) derivative underwent oxidation and dimerization that was extensive after

the solution was aerated with oxygen overnight (Figure 4.1). It should be noted that this

preparation contained a mixture of iso[4]LGE2-pyrrole unreacted iso[4]LGE2 and

dipeptide as well as byproducts derived from them, possibly including peracid generated

through oxidation of the aldehyde functional group in iso[4]LGE2.

Incubation of N-acetyl-gly-lys methyl ester with iso[4]LGE2 under an atmosphere of

argon, including bubbling argon gas before and during reaction, produced iso[4]LGE2-

pyrrole (4.3) as well as products of further oxidation, i.e., lactam, hydroxylactam. Sodium

dithionite (Na2S2O4) is an oxygen scavenger that can maintain the oxygen concentration

at extremely low levels.2 Only iso[4]LGE2-pyrrole (4.3) was produced if the adduction

reaction was carried out in water solution in the presence sodium dithionite under an

atmosphere of argon at 25 °C. An excess of acetyl-gly-lys methyl ester (20 equivalents)

was incubated with iso[4]LGE2 to favor complete consumption of the γ-ketoaldehyde.

Since sodium dithionite prevented oxidative transformations of the pyrrole when exposed

123

to air, it was essential to completely remove dithionite from the sample in order to study

further oxidative reactions. The reaction solution was concentrated under high vacuum to

remove water and methanol was added to dissolve the organic product. Then the mixture

was loaded onto a column of silica gel packed with a slurry in EtOAc and eluted with

methanol to remove most of sodium dithionite. After concentration under reduced

pressure, reagent grade chloroform that had been sparged with argon was added to

dissolve iso[4]LGE2-pyrrole and the solution was filtered to remove sodium dithionite

and any silical gel that had dissolved in the methanol. The crude iso[4]LGE2-pyrrole

solution was concentrated by evaporation with argon gas and re-suspended in 10%

acetonitrile/water for further use.

NH

O HN COOCH3

NH2O+

C

O

C5H11

OO

OH

OH Ar GasNa2S2O4

H2O as solvent

4.1 4.2

N CC5H11

OHO

HN

ONH

O COOCH3

OH4.3

Scheme 4.2 Synthesis of iso[4]LGE2-pyrrole.

Before and immediately after removing the sodium dithionite, no oxidation products

were detected by MALDI initially (Figures 4.1A and B). Upon exposure to air, within

one hour, it formed various oxidized derivatives, e.g., lactam, hydroxylactam, and

bispyrrole (Figure 4.1C). After bubbling O2 overnight, more oxidized products had been

generated, including aminal cross-link including bispyrrole as the major product (Figure

4.1D). Because this sample of iso[4]LGE2-pyrrole contained unreacted acetyl-gly-lys

methyl ester, the formation of peptide-peptide aminal cross-link presumably was a

consequence of the presence of excess N-acetyl-gly-lys methyl ester in the sample,

perhaps involving a reaction between the pyrrole and the nucleophilic side chain residue

of a second molecule of dipeptide.

124

Removal of dithionite

Oxidation

under Air1 h

Aeration overnight

Figure 4.1 MALDI-TOF MS analysis of iso[4]LGE2-pyrrole and oxidation products

generated upon short term exposure of the reaction mixture from iso[4]LGE2 with N-

acetylglycyllysine methyl ester in the presence of dithionite to air or longer term aeration

with oxygen before or after removal of dithionite.

Panel A: Reaction mixture from N-acetylglycyllysine methyl ester and iso[4]LGE2

(molar ratio = 20:1) in the presence sodium dithionite under an atmosphere of argon at 25

°C overnight. Panel B: Iso[4]LGE2-pyrrole immediately after removing the sodium

dithionite. Panel C: Reaction mixture of iso[4]LGE2-pyrrole after 1 h exposure to air.

Panel D: Reaction mixture of iso[4]LGE2-pyrrole after bubbling O2 overnight.

125

4.2.2 The oxidation of HPLC-purified iso[4]LGE2-pyrrole with air exhibits an

induction period.

A sample of iso[4]LGE2-pyrrole contaminated with excess N-acetyl-gly-lys methyl

ester and unreacted iso[4]LGE2 was purified by HPLC using an acetonitrile/water

gradient using LC-ESI to detect the components of interest by monitoring the appropriate

molecular weights in the extracted ion channels (Figure 4.2). The fraction eluting at 12

min contained pure iso[4]LGE2-pyrrole. The structural integrity of HPLC-purified

iso[4]LGE2-pyrrole was confirmed by MALDI-TOF MS (See appendix Figure 4.1S).

Iso[4]LGE2-pyrrole-H2O ion Extraction -- 558 (+1)

Iso[4]LGE2-pyrrole-H2O (558, +1)

Acetyl-Gly-Lys-O-Methyl Ester ion Extraction -- 260 (+1)

A

TIC

B

C

Iso[4]LGE2-pyrrole

Acetyl-gly-lys-O-methyl ester

LC monitoring for pyrrole

LC monitoring for dipeptide

MS of pyrrole

Figure 4.2 LC-ESI for the purification of iso[4]LGE2-pyrrole with selected ion recording

for iso[4]LGE2-pyrrole. A) TIC, B) LC monitoring and MS analysis for iso[4]LGE2-

pyrrole-H2O (m/z 558 (+ H+)), C) LC monitoring for N-acetyl-gly-lys methyl ester (m/z

260 (+ H+)).

MALDI and triple quadrupole mass spectrometry was used to confirm that purified

iso[4]LGE2-pyrrole was not contaminated with N-acetyl-gly-lys methyl ester or

126

iso[4]LGE2. The background of matrix for MALDI-TOF and solvent used in triple

quadrupole mass spectrometry was determined to not exhibit any peaks corresponding to

N-acetyl-gly-lys methyl ester or iso[4]LGE2 in the matrix or solvent (no m/z around 260

(+1 for N-acetyl-gly-lys methyl ester) or 353 (+1 for iso[4]LGE2)). Peaks not

corresponding to the matrix or solvent peaks could be detected upon analysis by MALDI

or triple quadrupole mass spectrometry of N-acetyl-gly-lys methyl ester or iso[4]LGE2

(see appendix Figure 4.2S).

The reaction time course (Figure 4.3) for incubation of pure iso[4]LGE2-pyrrole

under air at 25 °C in aqueous acetonitrile solution (10% acetonitrile/H2O) was monitored

for eight days (see appendix Figure 4.3S for details). MALDI-TOF mass spectra of pure

iso[4]LGE2-pyrrole and the reaction product mixture after exposure to air for eight days

are shown in Figure 4.4. Compared to iso[4]LGE2-pyrrole with un-reacted N-acetyl-gly-

lys methyl ester and iso[4]LGE2, pure iso[4]LGE2-pyrrole was much more stable under

air. During the first two days, almost no oxidized product was formed. At longer reaction

times, 1:1 adducts, i.e., lactam, hydroxylactam and bispyrrole became prominent. But, as

expected, no peptide-peptide aminal cross-link formed since excess N-acetyl-gly-lys

methyl ester and iso[4]LGE2 were not present.

The pure pyrrole obtained by preparative HPLC (Figure 4.2: LC-ESI for the

purification of iso[4]LGE2-pyrrole) was no longer prone toward oxidation or dimerization

when exposed to oxygen. The product evolution time course for the reaction of this

HPLC-purified iso[4]LGE2-pyrrole with oxygen exhibited an induction period followed

by rapid increase in the generation of all products (Figure 4.3).

127

0 day 2 days 4 days 6 days 8 days0

20

40

60

80

100Pe

rcen

t of T

otal

Time

Bispyrrole-H2O Lactam-H2O Hydroxylactam-2H2O Pyrrole-H2O

Time Course of Pure Iso[4]LGE2-pyrrole Reaction under Air

Figure 4.3 Time course of the oxidative reactions of pure iso[4]LGE2-pyrrole under air at

25 °C for 8 days. As shown in appendix Figure 4.3S, the observed peak intensity for

pyrrole-H2O is 100 (for each time point). Peak intensity of bispyrrole-H2O, lactam-H2O

and hydroxylactam-2H2O was zero for the first two days, and then gradually increased.

The total intensity is the sum of pyrrole-H2O, bispyrrole-H2O, lactam-H2O and

hydroxylactam-2H2O. The peak intensity of each product is divided by the total intensity

to provide the percent of total shown in the figure.

128

Figure 4.4 MALDI-TOF MS of pure iso[4]LGE2-pyrrole and its oxidation products:

lower trace = pure iso[4]LGE2 -pyrrole, upper trace = after incubation under air at 25 °C

for 8 days.

4.2.3 Reaction of pure iso[4]LGE2-pyrrole with Nα-acetylcysteine.

The possible role of nucleophilic thiols and amines in pyrrole-mediated cross-linking

of proteins was investigated in a model system. To test the possibility that nucleophilic

thiols can participate in isoLG-derived pyrrole-mediated cross-linking, pure iso[4]LGE2-

pyrrole was incubated in the presence of N-acetylcysteine under air. N-acetylcysteine

(4.4) was added to pure iso[4]LGE2-pyrrole (4.3) and incubated under air at 25 °C for

several days (Scheme 4.3).

129

N CC5H11

OHO

HN

ONH

O COOCH3

OH

HN COOH

O CH2SH

+

4.3 4.4

N CC5H11

OHO

HN

ONH

O COOCH3

OHSHN

COOHO

Air25oC

4.5

Scheme 4.3 Reaction of pure iso[4]LGE2-pyrrole with N-acetylcysteine.

Representative MALDI-TOF spectra of the reaction mixture are shown in Figure 4.5.

In the presence of N-acetylcysteine, incubation of iso[4]LGE2-pyrrole at neutral pH under

air showed no pyrrole-to-pyrrole cross-linking. Thus, the thiol-containing compound

inhibited the cross-linking of iso[4]LGE2-pyrrole (Figure 4.6). Instead a series of new

ions was produced. They corresponded to pyrrole cysteine adducts and a series

derivatives produced from them by the addition of one or more atoms of oxygen (Figure

4.7). The amount of these products increased with longer incubation. A thiol-pyrrole

conjugate was detected by MALDI-TOF MS that presumably incorporates a bond

between the pyrrole ring at C-2 to the sulfur atom of the N-acetylcysteine.3-4 Thus, thiols

inhibit the pyrrole dimerization by intercepting pyrrole cation radicals generated by

electron transfer from the pyrrole to oxygen (Scheme 4.4).

130

A B

Figure 4.5 MALDI-TOF MS spectra of pure iso[4]LGE2-pyrrole reaction with N-

acetylcysteine under air. Panel A: Reaction mixture of pure iso[4]LGE2-pyrrole and N-

acetylcysteine (molar ratio = 1:10) at 25 °C for 2 days. Panel B: Reaction mixture of pure

iso[4]LGE2-pyrrole and N-acetylcysteine (molar ratio = 1:10) at 25 °C for 8 days.

131

Figure 4.6 MALDI-TOF MS of pure iso[4]LGE2-pyrrole and its reaction with N-

acetylcysteine. Trace A = pure iso[4]LGE2-pyrrole incubation under air at 25 °C for 8

days. Trace B = pure iso[4]LGE2-pyrrole reaction with N-acetylcysteine under argon at

25 °C for 8 days. Trace C = pure iso[4]LGE2-pyrrole reaction with N-acetylcysteine

under argon in the presence of sodium dithionite at 25 °C for 8 days. Trace D = pure

iso[4]LGE2-pyrrole reaction with N-acetylcysteine under air at 25 °C for 8 days.

132

Figure 4.7 MALDI-TOF MS of pure iso[4]LGE2-pyrrole reaction with N-acetylcysteine

under air at 25 °C for 8 days. A series of peaks that differ in m/z by 16 show the addition

of oxygen atoms.

133

R1

R2

N

H3C

pyrrole

protein

R1

R2N

H3C


protein

R1

R2N

H3C

protein

O O +R1

R2

N

H3C

pyrrole

protein

O O

R1

R2N

H3C

proteinR1

R2N

H3C

protein

R1

R2

N

H3C

H

H

HO OH

pyrrole-pyrrole

HNCH

C

CH2

O

O

SH

O CH3R1

R2

N

H3C

protein

OO

HN CHCOO

CH2

S

OCH3

H

H

R1

R2

N

H3C

protein

NH

CHCOO

CH2

SO

H3C

pyrrole-cysteine crosslink

N-acetylcysteine

HO OH

protein

OO

pyrrole-cysteine

R1

R2

N

H3C

HN CHCOOH

CH2

S

OCH3

H

protein

H

HO OH

O O

m/z 721 (+H+)

m/z 719 (+H+)m/z 1149 (+H+)

Scheme 4.4 Possible electron transfer mechanism for pure iso[4]LGE2-pyrrole reaction

with N-acetylcysteine.

4.2.4 Initiation of the oxidative transformations of iso[4]LGE2-pyrrole.

Because the oxidative transformations of iso[4]LGE2-pyrrole (from which excess

dipeptide and isoLG had been removed by HPLC) exhibited a reaction time course

consistent with an autocatalytic mechanism, a search was initiated to discover possible

initiators. Pure iso[4]LGE2-pyrrole was incubated under air in the presence of various

134

potential initiators. The mass spectra of the resulting reaction product mixtures are

presented in the appendix.

(i) Stability of iso[4]LGE2-pyrrole in air in the presence of Nε-acetyl-L-lysine,

ethanolamine, L-lysine or β-amyloid peptide

Known components of the reaction mixture for the synthesis if isoLG-pyrroles were

tested first as well as analogues including Nε-acetyl-L-lysine, ethanolamine, L-lysine and

β-amyloid peptide. Pure iso[4]LGE2-pyrrole was comparably stable upon exposure to

these compounds without decomposing or forming oxidized adduct. Thus, Nε-acetyl-L-

lysine was added to pure iso[4]LGE2-pyrrole under air. After 2 days incubation, no

reaction was detected either with one equivalent of Ac-lysine or with a large excess

relative to pyrrole. Also the results were no different at pH=3 or 7. A representative mass

spectrum of the reaction product mixture is shown in appendix Figure 4.4S. Similarly,

after incubation for 2 days in the presence of 10 equivalents of L-lysine, pure

iso[4]LGE2-pyrrole underwent no reaction. Additional incubation of iso[4]LGE2-pyrrole

resulted in gradual consumption of the pyrrole, but no identifiable products were detected

(see appendix Figure 4.5S). Similarly, no reaction was detected after 2 days incubation of

pure iso[4]LGE2-pyrrole in the presence of 10 equivalents of β-amyloid peptide (see

appendix Figure 4.6S).

Incubation in the presence of ethanolamine resulted in decomposition, especially with

a large molar excess. Thus, a solution of 100 mM ethanolamine in sodium phosphate

buffer was adjusted pH to 7. Incubation of iso[4]LGE2-pyrrole under air in the presence

of one equivalent of ethanolamine resulted in gradual consumption of the pyrrole, but

some remained un-decomposed after 2 days (Figure 4.7S left). However, in the presence

135

of 10 equivalents of ethanolamine, the pyrrole decomposed completely within 2 h (Figure

4.7S right).

(ii) Stability of iso[4]LGE2-pyrrole in air in the presence of N-acetyl-gly-lys methyl

ester

Pure iso[4]LGE2-pyrrole was incubated under air in the presence of N-acetyl-gly-lys

methyl ester. Various conditions (N-acetyl-gly-lys methyl ester in H2O, pH 7; N-acetyl-

gly-lys methyl ester in phosphate buffer, pH 8.6; and N-acetyl-gly-lys methyl ester

acetate salt in H2O, pH 5.8; molar ratio from 0.1 to 10 equivalents to iso[4]LGE2-pyrrole)

were tested. The results were quite similar under all conditions. The oxidized pyrrole

product dehydrated hydroxylactam formed within 1 h and percentage increased with

continued incubation. But no aminal cross-link or bispyrrole was detected (see appendix

Figure 4.8S). These results contrasted with the lack of reaction observed upon incubation

of the pyrrole in the presence of Nε-acetyl-L-lysine, ethanolamine, L-lysine or β-amyloid

peptide. It was considered that commercial N-acetyl-gly-lys methyl ester may contain

some impurities that account for this discrepancy. To test this hypothesis, the commercial

sample of N-acetyl-gly-lys methyl ester was purified by HPLC using an

acetonitrile/water gradient. A fraction containing pure N-acetyl-gly-lys methyl ester was

isolated and confirmed with MALDI-TOF MS. Purified iso[4]LGE2-pyrrole was then

incubated under air at 25 °C for 6 days in the presence of HPLC-purified N-acetyl-gly-lys

methyl ester (Figure 4.9S). After incubation for several days, no oxidized products

formed.

(iii) The effects of vitamin C, vitamin E, H atom donor ((R)-(-)-2-(2,5-

dihydrophenyl)glycine), free radical initiator (2,2'-azobis(2-methylpropionamidine)

136

dihydrochloride), UV irradiation, rose bengal, methylene blue, Co2(CO)8,

H2O2/K2CO3, dimethyldioxirane, microwave irradiation, di-tert-butyl peroxide,

riboflavin (VB2), K3Fe(CN)6, FeCl3, K2S2O8 (persulfate) on the air stability of pure

iso[4]LGE2-pyrrole

The purified isoLG-pyrrole is unchanged after incubation in aqueous solution under

air for 2 days. In contrast, about half of the crude, unpurified pyrrole was transformed

into lactam, hydroxylactam, and bispyrrole under these same conditions. Furthermore, the

oxidative transformations of pure pyrrole exhibited a reaction time course suggestive of

autocatalysis (Figure 4.1 above). Therefore, various free radical initiators, H atom donors

and potential initiators of pyrrole oxidation were tested for their ability to trigger the

oxidation of purified iso[4]LGE2-pyrrole by air. In the presence of large amounts (0.1

equivalents) of vitamin C, vitamin E and 2,2'-azobis(2-methylpropionamidine)dihydro-

chloride, the purified pyrrole was completely consumed but no products could be

detected in the mass range of m/z 400-2000 after incubation overnight. In the presence of

smaller amounts (0.01 equivalents) of these additives, some pyrrole (about 20%)

remained un-reacted, but no products could be detected in the mass range of m/z 400-

2000 after incubation overnight (appendix Table 4.1S). Traces of metal ions are

especially likely to be present in aqueous buffers. Therefore, catalysis by traces of redox

active metal ions was investigated to test their ability to trigger the oxidation of purified

iso[4]LGE2-pyrrole by air. Fe(III) is known to promote polymerization of pyrroles

through single electron transfer (SET) that generates a pyrrole radical cation. Reoxidation

of the resulting Fe(II) by oxygen allows Fe(III) to function as a catalyst.5-7 Similar

reaction was performed by adding catalytic amount of metallic salt free radical initiators

137

to iso[4]LGE2-pyrrole. All of these potential catalysts caused rapid consumption of the

pyrrole but no traces of lactam, hydroxylactam, and bispyrrole were detected under the

reaction tested. Reaction details were summarized in appendix Table 4.1S (e).

To test the hypothesis that the product distribution from oxidative transformations of

LG/isoLG-derived pyrrole can be influenced by a hydrogen atom donor ((R)-(-)-2-(2,5-

dihydrophenyl)glycine), the pyrrole was incubated for 2 h in the presence of 5

equivalents of this hydrogen atom donor. The pyrrole was completely consumed but no

traces of lactam, hydroxylactam or bispyrrole were detected. With 1 equivalent, the

pyrrole was partially consumed in 2 h, and completely consumed after incubation over

night, but no traces of lactam, hydroxylactam or bispyrrole were detected. Incubation of

the pyrrole for 2 days in the presence of 0.2 equivalents of this hydrogen atom donor

resulted in no apparent reaction. The pyrrole was stable and no traces of lactam,

hydroxylactam or bispyrrole were detected.

4.2.5 Autoxidation of iso[4]LGE2-pyrrole promoted by APS/TEMED, TEMPO and

TMAO.

Potential catalysts that could promote pyrrole-pyrrole oxidative coupling and

oxidation to lactams and hydroxylactones were tested. Persulfate ion was reported to

promote pyrrole polymerization by SET that generates a pyrrole radical cation.6

Incubation of the isoLG-pyrrole derivative of N-acetyl-gly-lys methyl ester with 1-2

mol% ammonium persulfate (APS) and 0.5-1 mol% tetramethylethylenediamine

(TEMED) generated a product mixture containing small amounts of pyrole-pyrole dimer

138

and much greater quantities of lactam and hydroxylactam (Figure 4.8), but these products

were decomposed by treatment with more persulfate.

Since it was a free radical reaction, it was quite hard to control reaction to form one

major product. If a little more of the APS (over 5 mol% equivalent) and TEMED (over 2

mol% equivalent) were added, the pyrrole was completely consumed but no lactam,

hydroxylactam or bispyrrole were detected. The products were similar for reacting at 25

°C and 37 °C. Representative spectra are shown in appendix Figure 4.10-11S.


lower trace = pure iso[4]LGE2-pyrrole, upper trace = after exposure to 2 mol% APS and

1 mol% TEMED at 37 °C for 2 h.

139

Nitroxide, also known as amino-N-oxide, is a functional group which contains an N-

O bond and two C-N bonds. Nitroxide radicals, especially (2,2,6,6-tetramethylpiperidin-

1-yl)oxyl, have been used7 as stable persistent free radical initiators. It is the most widely

used nitroxide radical owing to its remarkable persistence.8-10

Since tetramethylpyrrolidine-N-oxide (TEMPO) could function as a SET oxidant11,

we tested its ability to promote isoLG-pyrrole dimerization. Treatment of dimer-free

pyrrole with 10 mol % of TEMPO at 37 °C for 3 h cleanly generated a product mixture

containing similar amounts of lactam and pyrrole-pyrrole dimer, and the products were

not degraded by longer incubation or use of larger mol% of TEMPO. Thus, pyrrole-

pyrrole cross-linking of isoLG-pyrrole was also facilitated by the stable free radical

tetramethylpyrrolidine-N-oxide (TEMPO) (Figure 4.9, see next page).

TEMPO hydroxylamine (TEMPOH) is formed by one electron reduction of TEMPO.

In air, TEMPOH can be readily oxidized back to TEMPO. The applications of TEMPO in

biochemistry often relate to its reduction to TEMPOH. In such systems, reduction of

TEMPO usually involves metal ions as shown in Scheme 4.5.12

NO

+ Fe (II) NOH

+ Fe (III)

Scheme 4.5 Reduction of TEMPO by ferrous iron.

TEMPO can be reduced to TEMPOH by metal ions. To neutralize any traces of metal

ions that might be present, EDTA was added to the reaction mixture. The same

autoxidation of iso[4]LGE2-pyrrole occurred in the presence of EDTA (one equivalent

relative to TEMPO) as in its absence. Apparently metal ion catalysis is not required for

140

TEMPO to promote oxidative reactions of iso[4]LGE2-pyrrole. Represented spectra are

shown in appendix Figure 4.14-15S.


lower trace = pure iso[4]LGE2-pyrrole, upper trace = after exposure to 10 mol% TEMPO

at 37 °C for 3 h.

Although trimethylamine-N-oxide (TMAO) is not a stable free radical analogue of

TEMPO, it has a nitrogen bound oxygen atom and can act as a prooxidant in the

autoxidation of methyl linoleate.12 TMAO can also react with Cu(I) and Cu(II)

derivatives to generate transient oxo-Cu(III) (cupryl) intermediates in oxidations such as

141

ortho hydroxylation of N-benzoyl-2-methylanaline.12-13 TMAO decomposes to aminium

radical TMA·+ then give the form of TMA (trimethylamine).14

Levels of TMAO are strongly positively correlated with cardiovascular disease risk.15

Understanding the molecular mechanisms of its ability to promote oxidative

transformations of biomolecules is likely to provide important insights into its

pathological involvements.

Remarkably, TMAO proved similarly effective to TEMPO in promoting oxidation of

the isoLG-pyrrole derivative of N-acetyl-gly-lys methyl ester to lactam and

hydroxylactam and oxidative pyrrole-pyrrole coupling. It is especially interesting that

TMAO can promote the oxidative transformations of isoLG-pyrroles. Treatment of

dimer-free pyrrole with 10 mol% of TMAO at 37 °C for 3 h cleanly generated a product

mixture containing similar amounts of lactam and pyrrole-pyrrole dimer, and the products

were not degraded by longer incubation or use of larger mol% of TMAO. All reactions

were done at 37 °C which was closed to human biological environment. We also

characterized this reaction including examining the role of trace metal ions through

adding EDTA (EDTA was equivalent to TMAO). Result indicated no direct effect to the

pyrrole initial reaction. Represented spectrums were shown in appendix Figure 4.12-13S.

It is conceivable that this chemistry contributes to the pathological consequences of

TMAO biosynthesis that involves gut microbial metabolism of phosphatidylcholines,16-17

and L-carnitine.18 Furthermore, exploration of the mechanisms of LG/isoLG pyrrole

oxidation reactions may provide insights into chemistry involved in pathologies

associated with elevated levels of TMAO.

142

R1

R2N

H3C

pyrrole

protein

R1

R2N

H3C


protein

R1

R2N

H3C

protein

O O +R1

R2N

H3C

pyrrole

protein

O O+

R1

R2N

H3C

proteinR1

R2N

H3C

protein

R1

R2N

H3C

proteinH

pyrrole

R1

R2N

H3C

protein

HL H

L

H

O OHO OH

pyrrole-pyrroleR1

R2N

H3C

protein

H

OHO H

R1

R2N

H3C

protein

HO

- H2O

R1

R2N

H3C

protein

H

Olactam

OO

NproteinR2

R1

R1

R2N

H3C

protein

OH

Ohydroxylactam

endoperoxide

O O

O

hydroperoxide

R1

R2N

H3C

protein

H

OO HL H

L

2H-pyrrol-1-ium

H

Scheme 4.6 Possible electron transfer mechanism for pyrrole oxidized reaction

The study results described above indicating the reaction of pure LG/isoLG-pyrroles

with oxygen is slow in the absence of catalysts. Nevertheless, it is likely that the first step

in the reaction of oxygen with these pyrroles generates a pyrrole cation radical-

superoxide ion pair. Hypothetical mechanistic pathways are presented in Scheme 4.6 for

the partitioning of this intermediate to generate pyrrole-pyrrole dimers, hydroxylactams

and lactams. Dimer formation involves addition of the electrophilic cation radical to the

electron rich pyrrole ring of a second molecule of pyrrole followed by further oxidation

and proton loss. Hydrogen peroxide is a likely byproduct of oxidative pyrrole-pyrrole

143

coupling. The tendency of isoLG pyrroles to undergo spontaneous oxidation may be the

result of a hydrogen peroxide promoted autocatalytic pyrrole oxidative coupling.

4.2.6 Pilot studies of bispyrrole synthesis.

Reaction conditions that promote bispyrrole formation were also probed with two

simple model systems, 3-ethyl-2,4-dimethylpyrrole and 3-ethyl-1,2,4-trimethylpyrrole.19

Pilot studies showed that both of these compounds formed dimers and trimers when

incubated under air in organic solvents. The details were discussed below and related

mass spectra of the reaction product mixtures were presented in appendix Figure 4.16-

18S.

(i) Initiate reactions of 3-ethyl-2,4-dimethylpyrrole with TEMPO and TMAO.

TEMPOor TMAO

NH

H3C CH3

CH3

HN

H3C

HN

CH3

CH3

CH3CH3

CH3

4.6 4.7

Scheme 4.7 Initiate reactions of 3-ethyl-2,4-dimethylpyrrole

Commercial available 3-ethyl-2,4-dimethylpyrrole (4.6) was resuspended in organic

solvent and tested its polymerization reactions in different conditions at 25 °C or 37 °C.

Pyrrole self-reaction in acetonitrile was tested first and result showed 3-ethyl-2,4-

dimethylpyrrole could form dimer and trimer spontaneously having no relationship with

adding TEMPO or TMAO (TEMPO or TMAO was 1:1 equivalent to pyrrole) (related

spectrums were shown in appendix Figure 4.16S). Methanol or acetone as solvents were

144

also tried and the results were quite similar. A possible way to avoid 3-ethyl-2,4-

dimethylpyrrole polemerization itself was pyrrole methylation with CH3I.

(ii) Preparation of 3-ethyl-1,2,4-trimethylpyrrole (MP)

NH

CH3I

KOH, DMSO N

4.6 4.8

Scheme 4.8 Preparation of 3-ethyl-1,2,4-trimethylpyrrole (MP)

Preparation of 3-ethyl-1,2,4-trimethylpyrrole 4.8 was carried out with the general

method Heaney and Ley developed.20 After reaction, combined organic extracts removed

solvent under reduced pressure to give pure 4.8 with silica gel column purification with

ethyl acetate: hexane=1:8 .

(iii) Initiate reactions of 3-ethyl-1,2,4-trimethylpyrrole with TEMPO and TMAO.

TEMPOor TMAO

N

H3C CH3

CH3

N

H3C

N

CH3

CH3

CH3CH3

CH3

4.8 4.9

Scheme 4.9 Initiate reactions of 3-ethyl-1,2,4-trimethylpyrrole with TEMPO and TMAO

3-Ethyl-1,2,4-trimethylpyrrole (4.8) was incubated under air at 25 °C in the presence

or absence of TEMPO (1 equivalent) in acetonitrile solution. 3-Ethyl-1,2,4-

trimethylpyrrole underwent dimerization and trimerization under both conditions (spectra

are shown in appendix Figure 4.18S), i.e, TEMPO had no significant effect on increasing

145

dimer formation. The relatively greater ease of autoxidation and pyrrole-pyrrole cross-

linking of pyrrole 4.8 compared to iso[4]LGE2-pyrrole could simply be a consequence of

the greater concentration of oxygen in organic versus aqueous medium. The

concentration O2 in air equilibrated H2O (0.28 mM) is about 7 fold lower than in air

equilibrated CHCl3 (2.05 mM), acetone (2.4 mM), acetonitrile (2.42 mM), or ethanol

(1.94 mM).21-27 Because the autoxidation of isoLG pyrrole was performed in the presence

of a lower concentration of O2 than autoxidation of 4.8, initiators, e.g., TEMPO or

TMAO exerted a greater influence on the autoxidation. Thus, autoxidation of isoLG

pyrrole occurs less readily than oxidation of 3-ethyl-1,2,4-trimethylpyrrole in air

saturated organic solvents in which the concentration of O2 is much higher than in water.

146

4.3 Conclusions

We discovered that the pure LG/isoLG-pyrrole derivative of the N-acetyl-lys-gly

methyl ester dipeptide can be produced in the presence of dithionite, an oxygen scavenger.

The oxidation and dimerization of the pure pyrrole is promoted by oxygen and exhibits

an induction period that can be eliminated by various catalysts, e.g., the stable free radical

TEMPO as well as by TMAO. More vigorous oxidative decomposition was promoted by

vitamin C, vitamin E and 2,2'-azobis(2-methylpropionamidine)dihydrochloride.

Previously, the reaction of phosphatidylethanolamine (PE) with an isoLG was reported to

produce a stable pyrrole dervative that resists further oxidation to lactams.28 This stability

was ascribed to lower concentrations of O2 in organic solvents such as CHCl3 (in which

the reaction of PE with isoLGs was conducted) than in H2O (the solvent used for the

reaction of proteins and the N-acetyl-lys-gly methyl ester dipeptide with isoLGs).

However, the opposite is the case, the concentration of oxygen in CHCl3 is seven times

higher than in air saturated H2O. Given the susceptibility of the autoxidation of the N-

acetyl-lys-gly methyl ester dipeptide-derived isoLG pyrrole to catalysis, the stability of

PE-isoLG pyrrole may be a consequence of its purity, i.e., the absence of catalytic

impurities that promote its autoxidation.

In the presence of N-acetylcysteine, incubation of iso[4]LGE2-pyrrole at neutral pH

under air showed no pyrrole-to-pyrrole cross-linking. Instead a series of new ions was

produced corresponding to pyrrole cysteine adducts and a series derivatives produced

from them by the addition of one or more atoms of oxygen. It is suggested that the thiol

inhibits the pyrrole dimerization by intercepting pyrrole cation radical generated by

electron transfer to oxygen.

147



following commercially available materials were used as received: acetyl-gly-lys-O-

methyl ester was from Bachem (Torrance, CA), 3-ethyl-2,4-dimethylpyrrole was from

Aldrich (Milwaukee, WI), all other chemicals were obtained from Fisher Scientific Co

(Chicago, IL). Iso[4]LGE2 was prepared by Dr. Jim Laird. Chromatography was

performed with ACS grade solvents.

4.4.2 Iso[4]LGE2-pyrrole. N-Acetyl-gly-lys methyl ester (2 mg, 7.71 µmol) was added

to 1 ml ddH2O and the solution was sparged with bubbled argon for several minutes to

remove air. Then Na2S2O4 (1 mg, 5.75 µmol) was added. Iso[4]LGE2 in methanol (136

µg, 0.386 µmol, 5 µg/µl) was added and the mixture was incubated overnight at room

temperature. TLC showed the presence of unreacted dipeptide, only one major product

and complete disappearance of iso[4]LGE2. The solution was concentrated under high

vacuum to remove water into a Dry Ice-acetone cooled trap, and then methanol (1 mL)

was added to dissolve the organic product. Then the mixture was loaded onto a 6 cm

column of silica gel packed with a slurry in EtOAc in a disposable pipette, and eluted

with methanol (2 mL) (Rf = 0.5) to remove most of sodium dithionite. After concentration

under reduced pressure, reagent grade chloroform (1 mL) that had been sparged with

argon was added to dissolve iso[4]LGE2-pyrrole and the solution was filtered to remove

sodium dithionite and any silical gel that had dissolved in the methanol. The crude

iso[4]LGE2-pyrrole (~222 µg, 0.39 µmol) solution was concentrated by evaporation with

argon gas and re-suspended in 500 µl 10% acetonitrile/water.

148

Iso[4]LGE2-pyrrole was purified by HPLC with a 2.1 x 100 mm i.d. C18 1.8 µm

column (Waters Acquity UPLC HSS). Chromatography was carried out with linear

elution gradient (elute A, 0.1% FA/H2O; elute B, 0.1% FA/ACN) at a flow rate of 150

µl/min. For determining the retention time of iso[4]LGE2-pyrrole (~222 µg, 0.39 µmol)

that had been freed of Na2S2O4 was dissolved in 10% acetonitrile (ACN) 0.1% formic

acid (FA) (50 µl) and then loaded to the column with 20% B for 10 min, and eluted with

the following gradient: 20-60% B over 20 min, to 98 % within 0.1 min and hold for 5 min.

The effluent was monitored by ESI-MS/MS with an API-3000 triple quadrupole

electrospray mass spectrometer (Applied Biosystems Inc.) The instrument was operated

in the positive mode and high-pressure nitrogen was used as source gas and scanning

from m/z 220-2000 using the parameters listed in Table 3.2. The pyrrole eluted between

12.8 and 14 minutes (Figure 4.2). Another aliquot of ~222 mg was injected and the eluent

was collected in 1 minute fractions from 11 minutes to 31 minutes and the fractions were

analyzed for purity by MALDI-TOF MS. Fractions containing the pure pyrrole were

combined. Optimized parameters for triple quadrupole mass spectrometer were noted in

section 3.4.4.

4.4.3 Autoxidation of crude iso[4]LGE2-pyrrole. Crude iso[4]LGE2-pyrrole that

contained excess dipeptide (~222 µg, 0.39 µmol in 500 µl 10% acetonitrile/water) was

incubated under air 1h on a shaker (IKA MTS 2/4 digital microtiter shaker, 500 rpm).

Then 100 µl of the reaction mixture (0.077 µmol) was quickly dried using a high-speed

vacuum evaporator and analyzed by MALDI-TOF MS. The remaining reaction mixture

149

(0.31 µmol) was aerated with oxygen overnight on a shaker (IKA MTS 2/4 digital

microtiter shaker, 500 rpm) at room temperature, and then analyzed by MALDI-TOF MS.

4.4.4 Autoxidation of iso[4]LGE2-pyrrole. Purified iso[4]LGE2-pyrrole (44 µg, 0.077

µmol in 450 µl 10% acetonitrile/water) was incubated under air at 25 °C on shaker (IKA

MTS 2/4 digital microtiter shaker, 500 rpm). At different time point (2, 6, and 8 days),

same amount of reaction mixture (15 µg, 0.026 µmol in 150 µl 10% acetonitrile/water)

was quickly dried using a high-speed vacuum evaporator and analyzed MALDI-TOF MS.

4.4.5 Autoxidation of iso[4]LGE2-pyrrole in the presence of Nα-acetylcysteine. 7.7 µl

Nα-acetylcysteine (0.77 µmol, 126 µg) (100 mM in H2O, pH 3) was added to purified

iso[4]LGE2-pyrrole (44.36 µg, 0.077 µmol in 500 µl 10% acetonitrile/water) and reacted

under air at 25 °C on shaker (IKA MTS 2/4 digital microtiter shaker, 500 rpm). At

different time point (2, 4, 6 and 8 days), same amount of reaction mixture (0.019 µmol,

125 µl) was quickly dried using a high-speed vacuum evaporator and analyzed by

MALDI-TOF MS.

Control experiments were performed. 7.7 µl Nα-acetylcysteine (0.77 µmol, 126 µg)

(100 mM in H2O, pH 3) was added to purified iso[4]LGE2-pyrrole (44.36 µg, 0.077 µmol

in 500 µl 10% acetonitrile/water) and reacted under argon gas protection at 25 °C on

shaker (IKA MTS 2/4 digital microtiter shaker, 500 rpm). At different time point (2, 4, 6

and 8 days), same amount of reaction mixture (0.019 µmol, 125 µl) was quickly dried

using a high-speed vacuum evaporator and analyzed by MALDI-TOF MS.

150

7.7 µl Nα-acetylcysteine (0.77 µmol, 126 µg) (100 mM in H2O, pH 3) was added to

purified iso[4]LGE2-pyrrole (44.36 µg, 0.077 µmol in 500 µl 10% acetonitrile/water) and

reacted under argon in the presence of dithionite at 25 °C on shaker (IKA MTS 2/4

digital microtiter shaker, 500 rpm). At different time point (2, 4, 6 and 8 days), same

amount of reaction mixture (0.019 µmol, 125 µl) was quickly dried using a high-speed

vacuum evaporator and analyzed by MALDI-TOF MS.

4.4.6 Autoxidation of iso[4]LGE2-pyrrole in the presence of acetyl-gly-lys methyl

ester. 7.7 µl N-acetyl-gly-lys methyl ester (0.77 µmol, 200 µg) (100 mM in H2O, pH 7)

was added to purified iso[4]LGE2-pyrrole (44 µg, 0.077 µmol in 500 µl 10%

acetonitrile/water) and reacted under air on shaker (IKA MTS 2/4 digital microtiter

shaker, 500 rpm). At different time point (1 h, 8 h, 1 and 2 days), same amount of

reaction mixture (0.019 µmol, 125 µl) was quickly dried using a high-speed vacuum

evaporator and analyzed by MALDI-TOF MS.

N-Acetyl-gly-lys methyl ester was purified by HPLC on a 2.1 x 100 mm i.d. C18 1.8

µm column (Waters Acquity UPLC HSS). N-Acetyl-gly-lys methyl ester (10 µl, 10

µmol, 259 µg) (100 mM in H2O, pH 7) was dissolved in 10 µl 2% ACN 0.1% FA. This

solution (18 µl) was injected onto the column and eluted with a linear gradient (elute A,

0.1% FA/H2O; elute B, 0.1% FA/ACN) at a flow rate of 100 µl/min. The gradient was 2-

8% B over 8 min. Fractions were collected every minute and analyzed for dipeptide by

MALDI-TOF MS. The fraction eluting during the 4th min contained purified N-acetyl-

gly-lys methyl ester. It was quickly dried using a high-speed vacuum evaporator and re-

suspended in 10% acetonitrile/water.

151

A mixture of purified N-acetyl-gly-lys methyl ester (0.77 µmol, 200 µg) and purified

iso[4]LGE2-pyrrole (44 µg, 0.077 µmol in 500 µl 10% acetonitrile/water) was incubated

under air at 25 °C for several days on shaker (IKA MTS 2/4 digital microtiter shaker, 500

rpm). At various time points (2 h, 6 h, 2 and 6 days) aliquots of the reaction mixture

(0.019 µmol, 125 µl) were quickly dried using a high-speed vacuum evaporator and

analyzed by MALDI-TOF MS.

4.4.7 Autoxidation of iso[4]LGE2-pyrrole in the presence of free radical initiators or

hydrogen atom donors. Vitamin C (7.7 µl of 1 mM in pH 7 H2O, 7.72 nmol, 1.36 µg)

was added to the purified iso[4]LGE2-pyrrole (44.36 µg, 0.077 µmol in 450 µl 10%

acetonitrile/water) and the mixture was incubated under air on shaker (IKA MTS 2/4

digital microtiter shaker, 500 rpm). At various time points (2 h, 24 h, 7 days), aliquots of

the reaction mixture (150 µl) were quickly dried using a high-speed vacuum evaporator

and analyzed by MALDI-TOF MS. Similar incubations were performed with 2,2'-

azobis(2-methylpropionamidine) dihydrochloride (1 mM in H2O, pH 6) or vitamin E (5

mM in EtOH, pH 6) in place of vitamin C.

(R)-(-)-2-(2,5-Dihydrophenyl)glycine (7.7 µl of 10 mM in 200 mM (pH 2) HCl,

0.077 µmol, 12 µg) was added to purified iso[4]LGE2-pyrrole (44 µg, 0.077 µmol in 450

µl 10% acetonitrile/water) and the mixture was incubated under air on shaker (IKA MTS

2/4 digital microtiter shaker, 500 rpm). At various time points (2 h, 24 h, 7 days), aliquots

of the reaction mixture (150 µl) were quickly dried using a high-speed vacuum

evaporator and analyzed by MALDI-TOF MS.

152

4.4.8 Autoxidation of iso[4]LGE2-pyrrole in the presence of APS/TEMED, TMAO

or TEMPO. Trimethylamine-N-oxide (TMAO, 7.7 µl of 1 mM in pH 7 H2O, 7.7 nmol,

5.8 µg) was added to purified iso[4]LGE2-pyrrole (44 µg, 0.077 µmol in 500 µl 10%

acetonitrile/water) and mixture was incubated under air on a shaker (IKA MTS 2/4 digital

microtiter shaker, 500 rpm). At various time points (1 h, 2 h, 3 h and 1 day), aliquots of

the reaction mixture (0.019 µmol, 125 µl) were quickly dried using a high-speed vacuum

evaporator and analyzed by MALDI-TOF MS. The same experiment was performed with

(2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO, 1 mM in 10% ACN, pH 6) in place of

TMAO.

Ammonium persulfate (APS, 1.6 µl of 1 mM in H2O, 1.544 nmol, 352.4 ng) and

tetramethylethylenediamine (TEMED, 0.7 µl of 1 mM in H2O, 0.772 nmol, 90 ng) were

added to iso[4]LGE2-pyrrole (44 µg, 0.077 µmol in 500 µl 10% acetonitrile/water) and

incubated under air on a shaker (IKA MTS 2/4 digital microtiter shaker, 500 rpm). At

various time points (1 h, 2 h, 3 h and 1 day) an aliquot of the reaction mixture (0.019

µmol, 125 µl) was quickly dried using a high-speed vacuum evaporator and analyzed by

MALDI-TOF MS.

4.4.9 3-Ethyl-1,2,4-trimethylpyrrole (4.8). Anhydrous DMSO (20 ml) was added to

crushed KOH pellets (2.24 g, 0.04 mol) and the mixture was stirred for 5 min. 2,4-

dimethyl-3-ethylpyrrole (4.6, 1.23 g, 0.01 mol) was added and the mixture was stirred for

45 min. The mixture was cooled in ice and CH3I (2.84 g, 0.02 mol) was added dropwise

with stirring and the stirring was continued for 45 min at room temperature. H2O (20 ml)

was added and the mixture was extracted with Et2O (3 × 50 ml). The combined organic

153

extracts were washed with H2O (3 × 25 ml), dried (MgSO4), and the solvent was removed

under reduced pressure. The residue was purified by flash chromatography (ethyl acetate:

hexane = 1:8, Rf = 0.8, yield 50%) to give 4.8 (0.68 g).29

TEMPO (10 µl of 100 mM in acetonitrile, 1 µmol, 156 µg) was added to 10 µl 3-

ethyl-1,2,4-trimethylpyrrole (1 µmol, 137 µg) (100 mM in acetonitrile) and the mixture

was incubated under air on a shaker (IKA MTS 2/4 digital microtiter shaker, 500 rpm) for

3 h. The reaction mixture was quickly dried using a high-speed vacuum evaporator and

analyzed by MALDI-TOF MS.

4.4.10 Analysis of adducts using MALDI-TOF MS. Initial diagnostic analysis of the

samples was performed by MALDI-TOF mass spectrometry (as described in section

3.4.3).

154

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158

CHAPTER 5

GENERATION OF AN ANTI-ISO[4]LGE2 MONOCLONAL ANTIBODY

5.1 Background

IsoLGs are generated through free radical-induced oxidation of arachidonates (see

chapter 1). They are extraordinarily reactive; forming covalent adducts incorporating

protein lysyl ε-amino groups within seconds. Since proteins so efficiently trap them, also

many plasma proteins have half-lives of several weeks, and modified proteins and

protein-protein cross-links are resistant to proteolysis,1 isoLGE2 adducted plasma proteins

are expected to accumulate in plasma which could provide a unique marker and effective

dosimeter for the oxidative injuries in the brain or retina.

Development and validation of isolevuglandin (isoLG) biomarkers of oxidative stress

could be used to assess the antioxidant effects of dietary supplements in vivo and to

examine their mechanisms of action, efficacy and effectiveness with respect to human

health. However, our understanding of these processes remains primitive, in part because

of the lack of sensitive and specific tools for detecting cumulative damage resulting from

oxidative stress. An enzyme-linked immunoassay (ELISA) with polyclonal antibodies

against isoLG-protein adducts reveals disease-related elevations in these biomarkers in

blood from patients with end-stage renal disease, cardiovascular disease, age-related

macular degeneration, and children born prematurely, as well as in brain tissue from

individuals with autism spectrum disease and liver from a murine model of alcoholic

liver disease.2 Therefore, detection of blood plasma isoLGE2 may provide a convenient,

minimally invasive method for monitoring such pathologies.

Previously, we generated anti-iso[4]LGE2 and anti-LGE2 polyclonal antibodies (pAb)

that have been useful for ELISA or immunohistochemistry for detection of iso[4]LGE2-

and LGE2-protein adducts.3 These antibodies are structurally specific showing low cross

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reactivity. They allow the confirmation that a variety of structural LG isomers are

produced in vivo. Specially, isoLGE2-KLH pAb bind isoLGE2-human serum albumin

(HSA) 200 times more strongly than they bind iso[4]LGE2-HSA, while the iso[4]LGE2-

KLH pAb bind iso[4]LGE2-human serum albumin (HSA) at least 2000 times more

strongly than they bind LGE2-HSA.4

However, pAbs have shortcomings that complicate standardization and accuracy.

They vary from batch to batch, resulting in variable responses, background can be high

and amounts are limited by the life-time of the animal source. In contrast, monoclonal

antibodies can be generated in unlimited quantities, and their specificity ensures that only

one epitope is recognized on an antigen. This is extremely useful when observing subtle

protein alterations, and the antibodies can be selected to exhibit low background cross

reaction, e.g., with abundant blood proteins such as serum albumin. Therefore, the

generation of a highly specific monoclonal anti-iso[4]LGE2 antibody was undertaken.

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5.2.1. Antibody production

Monoclonal anti-iso[4]LGE2 antibody was produced at the Hybridoma Cole Facility

of Visual Sciences Research Center (VSRC), Case Western Reserve University. Five

Balb/c mice were immunized with iso[4]LGE2-KLH. After 60 days, blood was draw from

each mouse, and tested for immune response. High anti-iso[4]LGE2 responses were

present in all 5 mice. Blood from mouse #972 showed high anti-iso[4]LGE2 response and

the lowest cross-reactivity to LGE2-BSA (Figure 5.1). Competitive antibody binding

inhibition studies with serum from all mice confirmed this conclusion (appendix Figure

5.1S). Therefore, mouse #972 was chosen for monoclonal antibody production.

Figure 5.1 Mouse serum immune responses (1:10,000 dilutions) comparison with pre-

immune serum.

Splenocytes from mouse 972 were fused with myeloma cells and clone screening was

done by both indirect and competitive ELISAs. Single cell clones from each round of

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subcloning that produced the anti-iso[4]LGE2 monoclonal antibody were identified, and

the clone exhibiting the strongest apparent immune-reactivity was grown in larger

amounts. Anti-iso[4]LGE2 mAb from this clone was purified as described below using

protein G column and the protein concentration of the purified mAbs was determined at

OD 280 nm.

From fusion attempted on 1920 samples (wells on 20 plates) 725 wells were found to

contain hybridoma cells (appendix Figure 5.2S). The antigens iso[4]LGE2-BSA and KLH

were used for the first screening tests. Of 371 large and very large clones (clones with

many cells), three showed strong response (clones number 9G8, 12G4, 18G7) against

iso[4]LGE2-BSA, and two others showed lower response (clones number 2E8, 15A5). Of

354 small and medium clones (clones with less cells), six (clones number 4G5, 5B7, 8B3,

10C11, 16E4, 19A4) showed lower response against iso[4]LGE2-BSA compared to the

above five clones from large clones (9G8, 12G4, 18G7, 2E8, 15A5). More antigens

(iso[4]LGE2-BSA, iso[4]LGE2-HSA, BSA, HSA, LGE2-BSA, LGE2-KLH, CEP-HSA)

were tested by indirect and competitive ELISAs of the supernatant of each positive clone

(appendix Figure 5.3S). Clones 9G8, 12G4, 18G7 showed the best responses. Clones 9G8

and 12G4 were chosen for the first round of subcloning. The isotype of these was

determined. Clone 9G8 was IgM and 12G4 was IgG1.

After the first screening, the chosen clones were selected by another two rounds of

subcloning to test clone cells’ productivity and consistency, and to confirm that the clone

was derived from a single cell. Since the isotype of 9G8 was IgM, we mainly focused on

clone 12G4 that was IgG1. Clone 12G4 had 34 first-round subclones and all 34 clones

showed strong response. It showed strong specificity to iso[4]LGE2-BSA and

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iso[4]LGE2-HSA and almost no response to LGE2-BSA (appendix Figure 5.4S). To

determine an optimal clone from 12G4 for the next round of subcloning, 10 clones were

chosen from the 34 (first subclones) for test again. Both the original antibody supernatant

concentration and a 10 fold dilution were tested. Both showed strong binding ability.

With more cell supernatant, competitive ELISA of these ten 12G4 clones was tested,

12G4 F3 was chosen for next round subcloning (appendix Figure 5.5S).

The supernatant and a 10 fold dilution from each of 16 secondary subclones of 12G4

F3 was tested by indirect ELISA (appendix Figure 5.6S). The final three candidates from

12G4 F3--C1 D1 G4 were chosen and fully tested by competitive ELISA (appendix

Figure 5.7S). Clone 12G4 F3 D1 was selected for adapting to serum free medium

(appendix Figure 5.8S).

Anti-iso[4]LGE2 mAb in serum free medium was diluted with binding buffer (20 mM

sodium phosphate buffer, pH 7) and loaded onto a protein-G column using commercially

available protein-G beads. Non-IgG proteins were washed away by eluting the column

with neutral binding buffer (20 mM sodium phosphate buffer, pH 7) and the

decomplexation is accomplished by changing the pH of the eluting buffer (glycine-HCl,

pH 2.6). The purified mAb solution was neutralized and dialyzed immediately against 10

mM PBS (pH 7.4) and then lyophilized and stored at -80 ℃. No non-specific binding was

observed in the purified mAb sample analyzed by sodium dodecyl sulfate polyacrylamide

gel electrophoresis (SDS-PAGE) as shown in Figure 5.2. Purification of IgG was

confirmed since only signal bands (heavy and light chain) were detected in SDS-PAGE.

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Heavy Chain

Light Chain

kD

Figure 5.2 SDS-PAGE analysis of purified anti-iso[4]LGE2 mAb with protein G column.

5.2.2. Specificity of anti-iso[4]LGE2 mAb.

To assess the structural specificity and selectivity of the monoclonal antibody,

competitive inhibition of antibody binding to iso[4]LGE2-modified proteins by various

haptens was examined. For all cross-reactivity studies, iso[4]LGE2-BSA was used as

coating agent and iso[4]LGE2-HSA was used as a standard. The concentration at 50%

inhibition (IC50) for iso[4]LGE2-HSA was defined as 100% cross-reactivity. Duplicates

of serial dilutions of all inhibitors were used and final curves were constructed using

mean absorbance values (Figure 5.3).

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Figure 5.3 Binding inhibition of protein G purified anti-iso[4]LGE2 mAb from serum

free medium by iso[4]LGE2-HSA, iso[4]LGE2-KLH, LGE2-BSA, LGE2-KLH, CEP-

BSA, CEP-HSA, BSA and HSA.

The IC50 values refer to the concentration of antigen that is required to inhibit 50%

binding of the antibody to the coating agent. The IC50 for iso[4]LGE2-HSA and

iso[4]LGE2-KLH using iso[4]LGE2-BSA as coating agent are 88.2 µM and 106.6 µM,

respectively. For a variety lysyl-ε-amino pyrrole protein modifications that have a

carboxylic acid group and are derived from lipid oxidation products (LGE2-BSA, LGE2-

KLH and CEP-BSA, CEP-HSA), anti-iso[4]LGE2 mAb showed remarkable selectivity.

All of them showed no significant cross-reactivity. Equally important, BSA and HSA

were not recognized at all by this antibody.

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5.2.3. Western analysis of anti-iso[4]LGE2 mAb.

The anti-iso[4]LGE2 mAb which exhibited high specificity towards the iso[4]LGE2

epitope, allow the development of immunochemical assays for the detection and

quantification of iso[4]LGE2 adducts generated under oxidative stress. It was of special

importance to employ these methods for diagnosing or screening for diseases in which

elevated levels of iso[4]LGE2 adducts were present at the disease site, or at remote sites,

as in the blood.

We did pilot western blot analysis against iso[4]LGE2-BSA, a standard protein

containing the iso[4]LGE2 modification plus BSA control. Different sample loading

amounts and primary concentrations were tested (Figure 5.4). Anti-iso[4]LGE2 mAb

showed both high selectivity and sensitivity.

Marker 10µg 5µg 1µg 0.1µg10ngIso[4]LGE2-BSA

5 µgBSA

1Ab: 10 µg/ml in PBS2Ab: 1:2000

Marker 5µg 1µg 0.1µg 10 ng


170130

95

72

55

43

34

26

17

Iso[4]LGE2-BSA

Marker5µg1µg0.1µg10 ng


Iso[4]LGE2-BSA

Figure 5.4 Comparative Western analyses using anti-iso[4]LGE2 mAb against

iso[4]LGE2-BSA, a standard protein containing the iso[4]LGE2 modification. Left panel:

developed with anti-iso[4]LGE2 mAb concentration as 10 µg/ml in PBS. Middle panel:

developed with anti-iso[4]LGE2 mAb concentration as 5 µg/ml. Right panel: developed

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with anti-iso[4]LGE2 mAb concentration as 1 µg/ml. Goat anti-mouse secondary

antibody was diluted 2000 times for all panel treatments.

A single-chain antibody from a phage displayed recombinant ScFv library that bound

a model peptide adducted with synthetic isoLGE2 was developed by Roberts et al.5

Recognition of isoLGE2–lysyl adducts isolated from oxidized arachidonic acid by this

anti-isoLGE2 adduct single-chain antibody was independent of the amino acid sequence

of the adducted peptides or proteins, and no cross-reactivity was detected with 4-

hydroxynonenal or 4-oxononanal adducts. But no test was performed to determine its

cross-reactivity with iso[n]LGE2-deived protein epitopes.5

The EC50 for oxidized arachidonyl–lysine adduct and isoLGE2-lysine adduct were

470 and 310 nM, respectively. HNE- or ONA-adducted peptides, and nonreactive F2-

isoprostane product, 15-F2t-IsoP (8-iso-PGF2a), showed no binding at peptide

concentrations up to 5 µM. These results indicated that single-chain antibody would

specifically recognize isoLGE2-lysine adducts, but not those formed by other pyrrole-

forming lipid aldehydes formed during free radical-mediated lipid peroxidation.5

Importantly, the binding of this antibody with iso[n]LGs was not determined.

Iso[4]LGE2 is an isomer formed exclusively through the free radical oxidation of AA-

PC, unlike isoLGE2 which can also be produced through the COX pathway.6 The anti-

iso[4]LGE2 mAb produced in this thesis research allows unambiguous assessment of the

formation of iso-LGs from the isoprostane pathway.

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5.3 Conclusions

An anti-iso[4]LGE2 mAb was prepared that exhibits high sensitivity and structural

specificity for detecting iso[4]LGE2–protein adduct epitope, and no significant cross-

reactivity with analogs including carboxyethylpyrrole (CEP) or LGE2-protein adduct

epitope that have carboxyalkyl side chains. The detection range of anti-iso[4]LGE2 mAb

for ELISA and western analysis was 5 µg/ml. Pilot Western blot analysis against

iso[4]LGE2-BSA, a standard protein containing the iso[4]LGE2 modification plus BSA

control, showed high specificity and selectivity with our anti-iso[4]LGE2 mAb.

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5.4.1 Materials. The following commercially available materials were used as received:

DMEM-high glucose (no pyruvate #10-017) was from Mediumtech. Oxaloacetic acid

(#O7753), insulin (#I4011), Na pyruvate (#P3662), 8-azaguanine (#A5284), hybridoma

growth supplement (NCTC-109) (#N1140), 50 x HAT (#H0262), 50 x HT (#H0137),

naphthol blue black (N3393) were from Sigma (St. Louis, MO). PEG (#783641) was

from Roche (Madison, WI). Fatal bovine serum (#16000-044), trypan blue (0.4%),

penicillin/streptomycin (PS #12800) and gentamicin (#15750-060) were from Gibco

(Grand Island, NY). CellTM mAb medium, quantum yield and animal component free

grade were from Becton&Dickinson (Franklin Lakes, NJ). HL-1TM chemically defined

serum-free medium was from LONZA. Protein G resin was from GenScript (Piscataway,

NJ). Glycine (161-0724) and Quick Start™ Bradford dye reagent (1x) were from Bio-

Rad (Hercules, CA). ABTS Ready to USE substrate solution was from Invitrogen.

Immunization stock iso[4]LGE2-KLH (3.604 μg/μl, pyrrole = 721.3 μM, ratio = 600.4)

was prepared by Dr. Jim Laird.

General methods:

(1) Macrophage extraction:

Materials used for macrophage extraction included 10 mL sterile syringes (one for

each mouse), 18 1x1/2 gauge needles (one for each mouse), 70% ethanol for

spraying, 2 autoclaved surgical scissors, forceps, curled fine forceps, 50 mL sterilized

conical tubes and an ice bucket. Macrophage medium, also called super HAT

medium, was prepared by reconstituting the contents of a commercial vial (medium

supplement (50×) Hybri-Max™ Sigma H0262, γ-irradiated, lyophilized powder) with

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10 mL of sterile cell culture medium (DMEM). The final working concentration of

each component was 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM

thymidine.

(2) SP2/0 myeloma cells growth:

Preparation of 8-azaguanine was achieved by reconstituting a commercial vial with

10 mL DMEM, to make a 50x stock; that then aliquoted as 2 mL per tube and stored

at -20 ℃. The final concentration was 130 µM. 100x Solution I stock was prepared by

adding 1320 mg oxaloacetic acid, 71 mg insulin (powder at 28 U/mg) and 550 mg Na

pyruvate to 100 mL PBS, then aliquoting as 6 mL per tube and stored at -20 ℃. The

final concentration of each component was 0.2 Units/ml insulin, 0.5 mM Na pyruvate

and 1 mM oxaloacetic acid. Solution I was a good supplement to DMEM medium to

provide the proper pH and enhance growth of the hybridoma cells. SP2/0 passage

medium contained 87 mL DMEM, 10 mL FBS, 1 mL 50x 8-azaguanine, 1 mL 100x

Solution I, 1 mL L-glutamine and 100 µl gentamicin. SP2/0 growth medium was

prepared by adding 20 mL FBS, 1 mL 100x Solution I, 2 mL NCTC-109, 1 mL L-

glutamine and 100 µl gentamicin to 78 mL DMEM.

(3) Fusion:

Materials used for fusion included sterile scissors, forceps, scalpels and sterile glass

slides with frosted end. Super DMEM medium was prepared by adding 120 mL FBS,

60 mL NCTC-109, 6 mL 100x Solution I, 12 mL 50x HAT or HT medium, 6 mL L-

glutamine and 600 µl gentamicin to 400 mL DMEM. Hybridoma medium (20%)

included 78 mL DMEM, 20 mL FBS, 6 mL NCTC-109, 1 mL 100x Solution I, 1 mL

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L-glutamine and 100 µl gentamicin. Hybridoma medium (15%) contained 83 mL

DMEM, 15 mL FBS, 2.5 mL NCTC-109, 1 mL 100x Solution I, 1 mL L-glutamine

and 100 µl gentamicin.

(4) Antibody purification:

Binding and elution buffers were 20 mM sodium phosphate buffer (pH 7) and 0.1 M

glycine, (pH 2.6) (375.35 mg/50 ml sterilized water, make fresh), respectively.

5.4.2 Immunization. Five ear tagged and numbered Balb/c mice were given

intraperitoneal (IP) injections of the immunogen (iso[4]LGE2-KLH). For the first IP

injection, 140 μl the antigenic stock solution (containing 500 μg of immunogen) was

emulsified with 360 μl of PBS and 500 μl FCA. Each mouse received 0.2 ml of the

emulsion utilizing a 25 gauge needle. The initial injection contained immunogen (100 μg

per mouse) in PBS and an equivalent volume of Fruend’s complete adjuvant (FCA,

Sigma F-5881). For booster IP injections, 70 μl the antigenic stock (containing 250 μg of

immunogen) solution was emulsified with 430 μl of PBS and 500 μl FIA. The animals

received their second injection two weeks after the first injection. The second injections

were given utilizing the same volume (0.2 ml) as the initial injection, but with half as

much immunogen (50 μg per mouse). Fruend’s incomplete adjuvant (FIA, Sigma, F-

5506) was used for this and all subsequent injections. Three weeks latter a third injection

was given and then the fourth injection. Ten to fourteen days following the fourth

injection a blood sample was collected from each mouse. The mice were anesthetized

with halothane at the Case animal facility. When the mice showed no response to the foot

pinch test, they were restrained by hand and the retro orbital sinus was punctured with the

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tip of a 250 μl non-heparinized capillary tube (Fisher cat# 02-668-10). Approximately

100 μl of blood was collected from each mouse. After the bleed, the mice were returned

to their respective cages to recover. Serum was prepared and tested from each blood

sample. Thus, collected blood was spun at 7000 rpm for 10 min. The serum was aspirated

and diluted 1:10,000 for ELISA analysis. Once a satisfactory immune response was

established in one of the mice, a fusion was initiated. Three days before the fusion, the

mouse with the best immune response was injected intravenously (IV. injection into the

tail vein) with 0.2 ml of the sterile antigenic solution (50 μg per mouse, 14 μl the

antigenic stock solution mixed with 186 μl of PBS) without adjuvant utilizing a 27 gauge

needle. The mouse was restrained with a small rodent restraint for this injection. For the

fusion, the mouse was euthanized via rapid cervical dislocation and the spleen aseptically

removed. The remaining four mice were kept until it was verified that antibody was

obtained that exhibited high specificity to the immunogen.

5.4.3 Cell counting. To determine the cell density and viability, cell counting was

performed using a hemocytomer. Thirty μl of the cell suspension was mixed with 30 μl

(equal volume) of 0.4% trypan blue solution to create a dilution factor of 2 and then

transferred into a fresh 1.5-mL tube followed by incubation for 5 min. With a cover-slip

in place, 10 μl of the trypan blue-stained cell suspension was loaded into a chamber on

the hemocytometer. All the cells were counted in the nine 1-mm wide center squares that

hold up to a total volume of 0.9 μl. Viable cells remained opaque, whereas non-viable

cells were stained in blue. The cell density and viability were calculated using the

following equations.

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Viable cell density (cells per mL) = (total number of viable cells/0.9μl) x dilution factor x

1000 μl/mL

Cell viability = (number of viable cells/number of viable and dead cells) x 100%

5.4.4 Macrophage intraperitoneal lavage exaction. Macrophage extraction from ten

Balb/c mice was performed two weeks before fusion. Another five mice were used for

each of two rounds of subcloning. After assembling all materials, autoclaving instruments

and preparing super HAT medium and storing it at 37 °C, mice were euthanized by rapid

cervical dislocation to avoid excessive bleeding into the peritoneal cavity. Thus, mice

were firmly held in one hand by the nape of neck and the tail was pulled with other hand

until a pop was felt owing to a separation between cranium and backbone. Mice were

sterilized with 70% ethanol and immediately placed into tissue culture hood. A small

incision was made for laparotomy in the abdominal skin of the mouse with forceps and

scissor. Mice were opened by pulling the skin firmly with both hands to each side so that

skin was completely pulled down. Then the peritoneum was sprayed with 70% ethanol. A

needle was uncapped inside the hood and 5-6 ml 11.6% of sucrose in sterilized water

(w/w) was squirted into the peritoneum. The needle was injected, hole-side down, into

the mouse in its lower abdominal area, preferably in a region where there was fat. During

the process, needle was inserted gently and not too deeply without perforating any organs

and the needle was withdrawn slowly so that the fat didn’t clog the needle. The mouse

skin was transparent after removing fur, so it could avoid puncture the organs when

inserted needle. The mouse was massaged the abdominal area to promote the detachment

of sucrose solution to tissues (15 seconds), and then repeated twice. For macrophage

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extraction, the needle of the same syringe was inserted horizontally into the upper part of

abdomen. Then about 4 ml sucrose solution containing macrophages was withdrawn.

This procedure was repeated twice to provide a total of about 12 ml fluid. The cell

suspension was poured into a 50 ml collection tube and the cells were centrifuged at 1000

rpm for 10 min at 20 °C. The cells were re-suspended using a 10 ml pipette and

macrophage medium was added to the pellet (10 ml per plate at a concentration of 105

cells/ml). The myeloma cells were fed with suspended macrophage cells, 100 µl per well

(104 cells/well) and then incubated at 37 °C in 5% CO2.

5.4.5 Growth of SP2/0 myeloma cells. The myeloma SP2/0 cells were grown in passage

medium (15% FBS) for about 6 days then slowly (after making sure that the cell viability

was 90% or higher before transfer to new medium, see p173 section 5.4.3) put into

growth medium (20% FBS). After centrifugation at 1200 rpm for 5 min at 4 °C, the

supernatant was removed and the pellet re-suspended in 10 ml of DMEM +

penicillin/streptomycin (PS) + 2% FBS.

5.4.6 Fusion Procedure. All fusion work was done in a cell culture laminar flow hood

under sterile conditions. The spleen was removed from the immunized mouse and placed

in a petri dish containing 10 ml of DMEM, penicillin/streptomycin (100 µl) and 2% FBS

(200 µl). The spleen was cut into 4 pieces. Each piece was gently ground between two

frosted end slides to release all cells from the connective tissue. The spleen cells were

collected in a 50 ml conical tube and put on ice. The SP2/0 myeloma cells were also

collected in a 50 ml conical tube and chilled on ice. Both sets of cells were washed twice

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with 20 ml of cold serum free-DMEM. Both pellets were re-suspended in 5 ml of cold

serum free-DMEM and put on ice. Using a hemocytometer, the spleen cells were counted

in a 20 μl aliquot and after adding 20 μl 4% acetic acid that would lyse any RBC’s.

Fusion was done at a cell ratio of 3:1 (spleen cells: myeloma). 6.3 million spleen cells

were used for this fusion, so the number of SP2/0 myeloma cells would be 2.1 million.

Using a hemocytometer, the myeloma cells were also counted in a 20 μl aliquot. 6.3

million spleen cells with 2.1 million myeloma cells were mixed in one 50 ml conical tube

and the mixed cells were spun down. All supernatant was removed and the pellet made as

dry as possible. The pellet was loosened by briskly tapping the tube. It was important to

break up the pellet so the PEG would contact as many cells as possible. The tube was

placed in a 37 °C water bath and 1 ml of 37 ℃ PEG 1500 solution (Ready-to-Use PEG

1500 (Cat#783641, 50% PEG 1500 (w/v) in 75 mM Hepes (pH 8.0)) from Roche) was

added dropwise directly onto the pellet over a full 1 minute period. The tube was rotated

while adding the PEG and gently swirled to resuspend the cells allowing the mixture to

sit in the water bath for 1 minute. While rotating the tube, 1 ml of 37 °C, serum free-

DMEM was added over 1 minute. Then 20 ml more of 37 °C serum free-DMEM was

added dropwise over 4 minutes. Then the cells were spun down at 1000 rpm for 5

minutes. It was not unusual to see two layers of cells at this stage. The supernatant was

removed immediately and the cell pellet was re-suspended in 100 ml super HAT medium.

Cell suspension (0.1 ml) was placed in each well of a twenty 96-well plate already that

contained macrophages, and the medium was changed every 5 days. By 10-14 days,

colonies of hybridoma cells were apparent. They usually grew on the periphery of each

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well. Culture supernatant (50 μl) could be removed for screening when 1/4 to 1/3 of the

well was covered with growing cells.

5.4.7 Clonal selection. At approximately 5 days post fusion the initial signs of viable

hybridoma colonies were observable in the wells. Concomitantly, the wells containing

unfused myeloma cells were dead or dying. By 12-14 days post fusion the hybridoma

colonies were macroscopically visible and ready for assay by ELISA for antibody

containing wells. After the first screening, viable clones were chosen and submitted to

another two rounds of subcloning to test the clone cells’ productivity and consistency,

and also to make sure that each clone was derived from a single cell.

5.4.8 Cultivation of hybridoma cells

The cultivation process was carried out inside a laminar flow hood. In order to prevent

cells from any potential contamination, all tools and containers were disinfected with

70% ethanol before using in the hood. A cryogenic vial containing 1 mL of frozen (in

liquid nitrogen) hybridoma cells (~107 cells) expressing anti-iso[4]LGE2 mAb was

thawed at 37 °C in a water bath immediately after being taken out of the liquid nitrogen.

The thawed cell suspension was transferred into 10 mL of cell culture medium DMEM in

a fresh 15-mL conical tube and spun down at 1000 rpm for 5 min at room temperature.

The supernatant was discarded after centrifugation. The cell pellet was resuspended

gently in 10 mL hybridoma medium (DMEM 8.3 mL, FBS 1.5 mL, NCTC-109 250 µl,

100x Solution I 100 µl, L-glutamine 100 µl and gentamicin 10 µl). The 10-mL cell

suspension was plated in a 100 mm cell culture dish with a seeding density of 106 viable

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cells/mL and a cell viability of 90% or higher (see p173 section 5.4.3) and incubated at

37 °C in 5% CO2. After 2-3 days, the cells were split into 3-4 fresh culture dishes with 10

mL of hybridoma medium for each. For freezing cell samples, about 108 hybridoma cells

(90% viable or higher) of each passage were harvested by centrifugation at 1000 rpm for

5 min and resuspended in 10 mL of freezing medium consisting of fresh DMEM (80%),

FBS (10%) and DMSO (10%). The cell suspension was transferred in 1-mL aliquots into

1.7-mL cryogenic vials that were later placed into a foam box, and subsequently frozen at

-80 °C for the first 24-48 hours, then placed into liquid nitrogen in a cryogenic tank for

long-term storage.

5.4.9 Adaptation of hybridoma cells to serum free medium

The hybridoma cells cultivated in hybridoma medium (mentioned above) were

sequentially adapted to HL-1TM chemically defined serum-free medium by following the

phases as indicated in Table 5.1. To avoid decreases of cell viability, the cells were

maintained under the same conditions in each phase for a few passages and then passaged

to the next phase until the cell viability reached to 90% or higher (see p173 section 5.4.3).

Table 5.1 Sequential adaptation of hybridoma cells to serum free medium

Phase Component of cell culture medium

1 100 % hybridoma medium

2 75% hybridoma medium; 25% HL-1 serum-free medium

3 50% hybridoma medium; 50% HL-1 serum-free medium

4 25% hybridoma medium; HL-1 serum-free medium

5 100% HL-1 serum-free medium

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5.4.10 Expression of anti-iso[4]LGE2 mAb

The hybridoma cells were subcultured with a seeding density of about 106 cells/mL in

200 mL of HL-1 serum-free medium with 2 mL of the penicillin and streptomycin

solution in two 250-mL shaking flasks. To maximize the production of anti-iso[4]LGE2

mAb, each flask containing 100 mL of cell culture was incubated at 37 °C in 5% CO2

with shaking at 60 rpm (Advanced digital orbital shaker from VWR) for 5-7 days without

changing the medium. By the end of this period, most of the cells died. The cell

suspension was transferred into six 50-mL conical tubes that were then centrifuged at 4

°C for 30 min at 10,000x g. The supernatant containing anti-iso[4]LGE2 mAb was

filtered through a 0.45 μm filter membrane (MILLEX® -GP syringe driven filter unit

from Millipore) with the aid of a water-driven aspirator, put into fresh tubes and kept at 4

°C.

5.4.11 Purification of the anti-iso[4]LGE2 mAb. A protein G column was packed with

immunopure protein G beads (5.0 ml), and was then equilibrated with 10 column

volumes of binding buffer (20 mM sodium phosphate buffer). Anti-iso[4]LGE2

monoclonal supernatant in animal component free medium (250 ml) was diluted 1:1 with

binding buffer (250 ml). 2-5 ml of Tris-HCl (pH 8) was added to adjust the pH>7 (pH is

important for IgG binding to protein G beads). The diluted supernatant was loaded onto

the top of the column and allowed to flow through it with gravity. In order to increase the

binding saturation of protein G, the eluate was recycled by loading it to columns three

times. In the future, it would be advisable to re-load the eluate to a washed column to see

if any mAb remained. Then binding buffer (50 ml) was passed through the column to

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remove unbound non-IgG proteins. IgG was then eluted with elution buffer (100 mM

glycine-HCl, pH 2.6) collected into 1.0 ml fractions containing 100 µl of 1 M Tris-HCl

(pH 9.5) buffer to neutralize the pH. The protein level of each eluted fraction was

qualitatively monitored using the Quick Start™ Bradford protein assay. Briefly, 100 μl of

an eluted fraction was added to 100 μl of Bradford dye reagent in a 96-well plate

followed by gently mixing with a pipette at room temperature. The color of the eluted

protein-containing solution (blue) was compared to the color of the reference solution

(bright green) containing a 1:1 mixture of elution buffer and Bradford dye reagent. The

more protein present in the solution, the more blue the mixture appeared to be. Collection

of fractions was terminated when the color of the dye mixture appeared nearly bright

green. The higher absorbance fractions (2-3 ml) were combined and dialyzed against 10

mM PBS (pH 7.4) 2×2 L at 4 °C for 24 h. The final concentration (1-2 mg/ml) was

measured at OD 280 nm. Several batches of mAb were purified, the concentrations

varies. The concentrations were 1.1 mg/ml, 1.13 mg/ml and 2.2 mg/ml for three different

batches. Binding buffer was used to re-equilibrate the column, then the column was

washed with 20% ethanol (15 ml) for storage at 4 °C.

5.4.12 Indirect enzyme-linked immunosorbent assay (ELISA) for anti-iso[4]LGE2

mouse serum, clone screening and purified mAb. Antigen (iso[4]LGE2-BSA,

iso[4]LGE2-HSA, BSA, HSA, LGE2-BSA, LGE2-KLH, CEP-HSA) solutions (1.2 μg in

10 mM PBS,100 μl/well, 0.012 μg/μl) was added to each well of a sterilized 96 well

ELISA plate (Cat. 9018, Corning Inc. Corning, NY). The plate was incubated overnight

at 4 °C. After the coating solution was discarded, each well was washed with PBST

180

(Tween 0.1%) (4×300 µl) and tapped onto paper towels until dry, then filled with 2%

BSA /PBS 300 µl, and incubated at 37 °C for 2 h to block the remaining active sites on

the plastic solid phase. In a pilot study, 3% chicken egg ovalbuman/PBS was tested as

blocking buffer. However, the blocking wasn’t efficient. The medium control also showed

a weak signal. Using 2% BSA/PBS as blocking buffer, the medium control showed

almost no signal, just like the untreated background. So 2% BSA/PBS was chosen as

blocking buffer. Each well was then washed 4 times with PBST (Tween 0.1%) and tapped

onto paper towels till dry. Mouse serum (100 μl diluted 10,000:1 with PBS) or clone

supernatant or protein G purified anti-iso[4]LGE2 mAb from HL-1 serum free medium (5

μg/ml in PBS) was dispensed into the ELISA plate wells and the plate was incubated

overnight at 4 °C. Pre-immune mouse serum (before injection with immunogen), diluted

as above, was employed as a negative response control if necessary. After discarding the

supernatant and washing with PBST (Tween 0.1%) (4×300 µl), 100 μl goat anti-mouse

HRP-conjugated secondary antibody (IgG specific), diluted 1:1200 with PBST (Tween

0.1%), was added to each well. Then the plate was incubated with shaking at room

temperature for 1 h using an orbital shaker (IKA MTS 2/4 digital microtiter shaker, 600

rpm). After discarding the supernatant, the wells were washed PBST (Tween 0.1%)

(4×300 µl). To each well was then added 100 µl of ABTS Ready-to-USE substrate

solution (from Invitrogen). The plate was allowed to develop at room temperature for 20

min at room temperature and all the absorbances were measured using a microplate

reader with a test wavelength of 450 nm.

5.4.13 Enzyme-linked immunosorbent assay (ELISA) for competitive antibody

181

binding inhibition studies using the anti-iso[4]LGE2 mAb. For antibody binding

inhibition studies to measure cross-reactivities, iso[4]LGE2-BSA was used as coating

reagent and iso[4]LGE2-HSA was used as a standard for purified anti-iso[4]LGE2 mAb.

LGE2-BSA, CEP-BSA, CEP-HSA, HSA and LGE2-KLH served as inhibitors. For each

inhibitor, up to eleven serial dilutions of a standard, a blank, and a positive control, were

run. Each well of a 96 well ELISA plate was coated with iso[4]LGE2-BSA solution (100

µl), prepared by diluting a solution containing 1016 μM iso[4]LGE2-BSA to 1.12 μM

(0.3 μg/well) with pH 7.4 PBS (10 mM). The plate was incubated overnight at 4 °C, then

washed with 10 mM PBST (Tween 0.1%) (4×300 µl) and then blocked by incubating 1 h

at 37 °C with 300 µl of 2% BSA in 10 mM PBS. Then the plate was rinsed with 10 mM

PBST (Tween 0.1%) (4×300 µl). Eleven serial dilutions of inhibitor or standard (120 µl

each with a dilution factor of 0.2) were pre-incubated at 37 °C for 1 h with protein G

purified anti-iso[4]LGE2 monoclonal antibody from HL-1 serum free medium (120 µl, 5

μg/ml in 10 mM PBS).The initial inhibitor and standard concentrations were iso[4]LGE2-

HSA (446.5 μM), iso[4]LGE2-KLH (446.5 μM), LGE2-BSA (446.5 μM), CEP-BSA

(446.5 μM), CEP-HSA (446.5 μM), BSA (446.5 μM) and HSA (446.5 μM) respectively.

These were prepared by diluting a iso[4]LGE2-HSA (893.3 μM), iso[4]LGE2-KLH (721.3

μM), LGE2-BSA (1974 μM), CEP-BSA (496 μM), CEP-HSA (623.6 μM), BSA (1500

μM) and HSA (1500 μM) with 10 mM PBS, respectively. Blank wells were filled with

2% BSA/PBS (100 µl). Positive control wells were filled with the diluted antibody

solution (50 µl) and PBS (50 µl). The antibody antigen complex solutions (100 µl) were

then added in duplicate to their respective halves of the plate, which was then incubated

at room temperature with gentle agitation on an orbital shaker (IKA MTS 2/4 digital

182

microtiter shaker, 600 rpm) for 1 h. After discarding the supernatant and washing with

PBST (Tween 0.1%) (4×300 µl), 100 μl goat anti-mouse HRP-conjugated secondary

antibody (IgG specific), diluted 1:1200 with PBST (Tween 0.1%), was added to each

well and then the plate was incubated with shaking at room temperature for 1 h on the

orbital shaker. After discarding the supernatant, the wells were washed PBST (Tween

0.1%) (4×300 µl). To each well was then added 100 µl of Ready-to-Use substrate

solution ABTS. The plate was allowed to develop at room temperature for 20 min at

room temperature and all the absorbances were measured using a microplate reader with

a test wavelength of 450 nm. The absorbance in each well was measured with a dual-

wavelength Bio-Rad 450 microplate reader (Hercules, CA) with detection at 405 nm

relative to 655 nm. Absorbance values for duplicate assays were averaged and scaled to

make the maximum curve fit value close to 100 percent. The averaged scaled percent

absorbance values were plotted against the log of concentration. Theoretical curves for

each plot were fit to the absorbance data with a four parameter logistic function, f(x) = (a-

d)/[1+(x/c)b]+d using Origin 9.0 (OriginLab Corporation, Northampton, MA). Parameter

a = the asymptotic maximum absorbance, b = slope at the inflection point, c = the

inhibitor concentration at the 50 % absorbance value (IC50), and d = the asymptotic

minimum absorbance.

5.4.14 Western analysis. It was carried out according to published protocols.7-8

Iso[4]LGE2-BSA (~ 10 ng -10 µg) was dissolved in Laemmli sample buffer,9 fractionated

on a 4-12% SDS-PAGE, and electro blotted onto a polyvinyl difluoride (PVDF)

membrane (Millipore, Bedford, MA). The PVDF membranes were then treated with

183

Odyssey blocking buffer (Li-Cor cat# 927-40000) and probed using anti-iso[4]LGE2

mAb at various dilutions. Goat anti-mouse IRDye® 680 (Odyssey cat# 926-32220) was

used as secondary antibody. The immunoreactive bands were tested and quantified using

a Li-Cor infrared imaging system.

184


5.5 References

1. Shringarpure, R.; Grune, T.; Sitte, N.; Davies, K. J. (2000) 4-Hydroxynonenal-

modified amyloid-beta peptide inhibits the proteasome: possible importance in

Alzheimer's disease. Cell Mol Life Sci 57 (12), 1802-1809.

2. Nagata, K.; Suzuki, H.; Sakaguchi, S. (2007) Common pathogenic mechanism in

development progression of liver injury caused by non-alcoholic or alcoholic

steatohepatitis. J Toxicol Sci 32 (5), 453-468.

3. Salomon, R. G.; Batyreva, E.; Kaur, K.; Sprecher, D. L.; Schreiber, M. J.; Crabb,

J. W.; Penn, M. S.; DiCorletoe, A. M.; Hazen, S. L.; Podrez, E. A. (2000) Isolevuglandin-

protein adducts in humans: products of free radical-induced lipid oxidation through the

isoprostane pathway. Biochim Biophys Acta 1485 (2-3), 225-235.

4. Salomon, R. G.; Sha, W.; Brame, C.; Kaur, K.; Subbanagounder, G.; O'Neil, J.;

Hoff, H. F.; Roberts, L. J. (1999) Protein adducts of iso 4 levuglandin E-2, a product of

the isoprostane pathway, in oxidized low density lipoprotein. J Biol Chem 274 (29),

20271-20280.

5. Davies, S. S.; Talati, M.; Wang, X.; Mernaugh, R. L.; Amarnath, V.; Fessel, J.;

Meyrick, B. O.; Sheller, J.; Roberts, L. J., 2nd (2004) Localization of isoketal adducts in

vivo using a single-chain antibody. Free Radic Biol Med 36 (9), 1163-1174.

6. Morrow, J. D.; Hill, K. E.; Burk, R. F.; Nammour, T. M.; Badr, K. F.; Roberts, L.

J., 2nd (1990) A series of prostaglandin F2-like compounds are produced in vivo in

humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc Natl Acad Sci

U S A 87 (23), 9383-9387.

185

7. Crabb, J. W.; Miyagi, M.; Gu, X. R.; Shadrach, K.; West, K. A.; Sakaguchi, H.;

Kamei, M.; Hasan, A.; Yan, L.; Rayborn, M. E.; Salomon, R. G.; Hollyfield, J. G. (2002)

Drusen proteome analysis: An approach to the etiology of age-related macular


8. Bhattacharya, S. K.; Rockwood, E. J.; Smith, S. D.; Bonilha, V. L.; Crabb, J. S.;

Kuchtey, R. W.; Robertson, N. G.; Peachey, N. S.; Morton, C. C.; Crabb, J. W. (2005)

Proteomics reveal cochlin deposits associated with glaucomatous trabecular meshwork. J

Biol Chem 280 (7), 6080-6084.

9. Laemmli, U. K. (1970) Cleavage of structural proteins during assembly of head of

bacteriophage-T4. Nature 227 (5259), 680-685.

186

Appendix

Adduct Form Formula Mass (Cal.) Pyrrole C20H28O3 316.20 Pyrrole monodehydrated C20H26O2 298.19 Lactam C20H28O4 332.20 Lactam monodehydrated C20H26O3 314.19 Hydroxylactam C20H26O5 348.19 Hydroxylactam monodehydrated C20H26O4 330.18 Hydroxylactam bisdehydrated C20H24O3 312.17

Table 2.1S Lysine modifications with iso[4]LGE2 Peptide Sequence m/z (obs) Mr (obs) ion score Adduct Modification form SVTGAK280QVNY 690.8754 (+2) 1379.7508 29 C20H26O3 Lactam Monodehydrated Table 2.2S Peptide of calpain-1 modified by iso[4]LGE2, as identified by Mascot analysis # b b++ b* b*++ b0 b0++ Seq. y y++ y* y*++ y0 y0++ #

1 88.0393 44.5233 70.0287 35.5180 S 10

2 187.1077 94.0575 169.0972 85.0522 V 1293.7089 647.3581 1276.6824 638.8448 1275.6984 638.3528 9

3 288.1554 144.5813 270.1448 135.5761 T 1194.6405 597.8239 1177.6140 589.3106 1176.6299 588.8186 8

4 345.1769 173.0921 327.1663 164.0868 G 1093.5928 547.3001 1076.5663 538.7868 7

5 416.2140 208.6106 398.2034 199.6053 A 1036.5714 518.7893 1019.5448 510.2760 6

6 858.4971 429.7522 841.4706 421.2389 840.4866 420.7469 K 965.5342 483.2708 948.5077 474.7575 5

7 986.5557 493.7815 969.5292 485.2682 968.5451 484.7762 Q 523.2511 262.1292 506.2245 253.6159 4

8 1085.6241 543.3157 1068.5976 534.8024 1067.6136 534.3104 V 395.1925 198.0999 378.1660 189.5866 3

9 1199.6671 600.3372 1182.6405 591.8239 1181.6565 591.3319 N 296.1241 148.5657 279.0975 140.0524 2

10 Y 182.0812 91.5442 1

Table 2.3S Mascot characterization of the doubly charged ion m/z 690.88 (SVTGAKQVNY) showed series of fragment ions sufficient to unambiguously identify a iso[4]LGE2 modification on the calpain-1 lysyl residue (K280).

Figure 2.1S Domain architecture of calpain-1 and three active sites (left panel). Minicalpain was shown as subdomain IIa and domain IIb, with catalytic triad residue Cys 115, His 272 and Asn 296 (right panel).

188

101.587.5

52.7

35.8

27.9

18.8

Catalyitc Subunit

Small Subunit

Control

Crosslink

2 h Figure 2.2S Coomassie blue staining image of calpain-1 after 2 h treatment with 460 equivalents of iso[4]LGE2

Crosslink part


Control one

Modified Calpain-1

Use Anti-Calpain1 catalyticsubunit primary antibody

30 min 1 h 3 h


ControlModified Calpain-1


30 min 1 h 3 h

Crosslink part

240 kDa

320 kDa

250

150

100

75

50

37

25

20

Use Anti-Iso[4]LGE2monoclonal antibody

Modified Calpain-130 min 1 h 3 h

Crosslink part


Control

250

150

100

75

50

37

2520

Figure 2.3S Western blot images of calpain-1 after 30 min, 1 h and 3 h treatment with 460 equivalents of iso[4]LGE2

189

Use Anti-Calpain 1 catalyticsubunit primary antibody

overnight

1 h 3 hcontrol overnight

overnight

1 h 3 h control


50 times 100 times

240 kDa160 kDa

320 kDa

120 kDa110 kDa

250

150

100

75

50

37

25

20


overnight

1 h 3 hcontrol overnight

overnight

1 h 3 h control

50 times 100 times




overnight

1 h 3 h overnight

overnight

1 h 3 h

50 times 100 times

control




160 kDa240 kDa

320 kDa

Use Anti-Iso[4]LGE2 monoclonal antibody

250

150

100

75

5037

2520

Figure 2.4S Western blot images of calpain-1 after 1 h, 3 h and overnight treatment with 50 and 100 equivalents of iso[4]LGE2

190

overnight

1 h3 h control6 hovernight

1 h3 h6 h

10 times 5 times


120 kDa110 kDa

160 kDa240 kDa

Use Anti-Calpain I catalyticsubunit primary antibody

overnight

1 h3 h control6 hovernight

1 h3 h6 h10 times 5 times




Use Anti-Calpain I smallsubunit primary antibody

Figure 2.5S Western blot images of calpain-1 after 1 h, 3 h, 6h and overnight treatment with 5 and 10 equivalents of iso[4]LGE2

Control2times 24h

2times 48h

5times 24h

5times 48h

10times 24h

10times 48h

20times 24h

20times 48h


120 kDa110 kDa

160 kDa240 kDa

Use Anti-Calpain I catalyticsubunit primary antibody

Control2times

24h2times 48h

5times 24h

5times 48h

10times 24h

10times 48h

20times 24h

20times 48h




Use Anti-Calpain I smallsubunit primary antibody

Figure 2.6S Western blot images of calpain-1 after 24 h and 48 h treatment with 2, 5, 10 and 20 equivalents of iso[4]LGE2

191

A1 A2

B1 B2

Figure 2.7S ESI-MS of synthesis fluorescence labeled iso[4]LGE2 intermediate (2.3, panel A1 and A2) and final product (2.4, panel A1 and A2) in both negative mode and positive mode. Adduct Form Formula Mass (Cal.) Pyrrole C30H41N5O5S 583.28 Pyrrole monodehydrated C30H39N5O4S 565.27 Lactam C30H41N5O6S 599.28 Lactam monodehydrated C30H39N5O5S 581.27 Hydroxylactam C30H41N5O7S 615.27 Hydroxylactam monodehydrated C30H39N5O6S 597.26 Hydroxylactam bisdehydrated C30H37N5O5S 579.25 Table 2.4S Lysine modifications with fluorescence labeled iso[4]LGE2.

192

C

OH

ONHCH2CH2NH

NO

N

SO2NCH3

CH3N

C

OH

ONHCH2CH2NH

NO

N

SO2NCH3

CH3N

O

Protein Lys

LysProtein

C

OH

ONHCH2CH2NH

NO

N

SO2NCH3

CH3N

O

LysProtein

HO

C5H11

C5H11

C5H11

H2O

CO

NHCH2CH2NH

NO

N

SO2NCH3

CH3NProtein Lys C5H11

Pyrrole with fluorescence tag

Pyrrole monodehydrated

H2O

Lactam monodehydrated

CO

NHCH2CH2NH

NO

N

SO2NCH3

CH3N

O

LysProtein C5H11

Lactam with fluorescence tag

Hydroxylactam with fluorescence tag

CO

NHCH2CH2NH

NO

N

SO2NCH3

CH3N

O

LysProtein

HO

C5H11

H2O

Hydroxylactam monodehydrated

H2O

CO

NHCH2CH2NH

NO

N

SO2NCH3

CH3N

O

LysProtein C5H11

Hydroxylactam bisdehydrated

Scheme 2.1S Chemical structures of Lys-fluorescence labeled iso[4]LGE2 adducts.

193

Table A b b++ b* Seq. y y++ y* #

1 114.0913 57.5493 L 6

2 243.1339 122.0706 E 1127.5918 564.2995 1110.5654 5

3 952.4961 476.7517 935.4695 K 998.5492 499.7782 981.5223 4

4 1023.5332 512.2702 1006.5067 A 289.1870 145.0972 272.1608 3

5 1094.5703 547.7888 1077.5438 A 218.1499 109.5786 201.1236 2

6 K 147.1128 74.0600 130.0867 1

Table b b b++ b* b*++ Seq. y y++ y* y*++ #

1 100.0757 50.5415 V 10

2 807.4222 404.2147 790.3956 395.7015 K 1512.7780 756.8926 1495.7515 748.3794 9

3 906.4906 453.7489 889.4641 445.2357 V 805.4315 403.2194 788.4050 394.7061 8

4 963.5121 482.2597 946.4855 473.7464 G 706.3631 353.6852 689.3365 345.1719 7

5 1062.5805 531.7939 1045.5539 523.2806 V 649.3416 325.1745 632.3151 316.6612 6

6 1176.6234 588.8153 1159.5969 580.3021 N 550.2732 275.6402 533.2467 267.1270 5

7 1233.6449 617.3261 1216.6183 608.8128 G 436.2303 218.6188 419.2037 210.1055 4

8 1380.7133 690.7133 1363.6867 682.3470 F 379.2088 190.1081 362.1823 181.5948 3

9 1437.7348 719.3710 1420.7082 710.8577 G 232.1404 116.5738 215.1139 108.0606 2

10 R 175.1190 88.0631 158.0924 79.5498 1

Table 2.5S Mascot characterization of the doubly charged ion m/z 620.86 (LEKAAK) and triply charged ion m/z 537.96 (VKVGVNGFGR) showed series of fragment ions sufficient to unambiguously identify a fluorescence labeled iso[4]LGE2 modification on the GAPDH lysyl residue.

194

Panel A:

Panel B:

Figure 2.8S The peak area of the amino acids was compared with the peak area of the quantitative standard amino acids (Standard H). The detection limit was tested by 25, 50, 100, 250, 400 and 500 pmol of standard amino acids. Reliable relative standard deviation (< 10%) was achieved and the detection limit was determined with standard amino acids up to 50 pmol and was plotted with all amino acids in (B).

195

Table 2.6S (1) Amino acid analysis template.

196

NAME/ID# GAPDH(Rabbit) Dimer Injection Id: 1489

VOL HYDROLYZED 20.00

RATIO APPLIED 0.01600

MOLECULAR WEIGHT 35780.00

AMINO ACID Known Composition pmol analyzed pmol protein over 15% under 15% exp comp integer comp % int err

His (H) 11.00 4.5 0.41 0.41 0.41 10.42 10.00 9.09

Asn (N) 17.00 0.00 FALSE

Ser (S) 19.00 13.28 0.70 FALSE FALSE 30.74 31.00 63.16

Gln (Q) 6.00 0.00 FALSE

Arg (R) 10.00 4.91 0.49 0.49 0.49 11.37 11.00 10.00

Gly (G) 32.00 23.94 0.75 FALSE FALSE 55.42 55.00 71.88

Asp (D) 20.00 10.7 0.54 FALSE FALSE 24.77 25.00 25.00

Glu (E) 13.00 6.31 0.49 0.49 0.49 14.61 15.00 15.38

Thr (T) 21.00 6.66 0.32 0.32 FALSE 15.42 15.00 28.57

Ala (A) 34.00 11.87 0.35 0.35 FALSE 27.48 27.00 20.59

Pro (P) 11.00 4.82 0.44 0.44 0.44 11.16 11.00 0.00

Lys (K) 26.00 6.81 0.26 0.26 FALSE 15.76 16.00 38.46

Tyrb (Y) 9.00 3.61 0.40 0.40 0.40 8.36 8.00 11.11

Met (M) 10.00 2.54 0.25 0.25 FALSE 5.88 6.00 40.00

Val (V) 35.00 9.86 0.28 0.28 FALSE 22.82 23.00 34.29

Ile (I) 20.00 6.17 0.31 0.31 FALSE 14.28 14.00 30.00

Leu (L) 18.00 9.2 0.51 FALSE FALSE 21.30 21.00 16.67

Phe (F) 14.00 5.14 0.37 0.37 0.37 11.90 12.00 14.29

TOTAL #AA 326.00 NEW ESTIMATE

PMOL PROTEIN AVERAGE %

ESTIMATED AMOUNT WITHIN 15% 0.43 ERROR 26.78

PMOL PROTEIN 0.43

15% OF PROTEIN 0.06 TOTAL PMOL AMINO ACID 130.32

-15% OF PROTEIN 0.36 TOTAL PMOL HYDROL 27.00 TOTAL UGRAMS 0.97

+15% OF PROTEIN 0.49 CONC pmol/ul 1.35 CONC ug/ul 0.05

Table 2.6S (2) Amino acid composition of dimer of modified GAPDH.

197

NAME/ID# GAPDH(Rabbit) Trimer Injection Id:

1494





His (H) 11.00 5.46 0.50 0.50 0.50 11.38 11.00 0.00

Asn (N) 17.00 0.00 FALSE

Ser (S) 19.00 13.29 0.70 FALSE FALSE 27.69 28.00 47.37

Gln (Q) 6.00 0.00 FALSE

Arg (R) 10.00 5.96 0.60 FALSE FALSE 12.42 12.00 20.00

Gly (G) 32.00 22.87 0.71 FALSE FALSE 47.65 48.00 50.00

Asp (D) 20.00 11.69 0.58 FALSE FALSE 24.36 24.00 20.00

Glu (E) 13.00 5.77 0.44 0.44 0.44 12.02 12.00 7.69

Thr (T) 21.00 8.72 0.42 0.42 0.42 18.17 18.00 14.29

Ala (A) 34.00 13.33 0.39 0.39 FALSE 27.78 28.00 17.65

Pro (P) 11.00 5.47 0.50 0.50 0.50 11.40 11.00 0.00

Lys (K) 26.00 7.52 0.29 0.29 FALSE 15.67 16.00 38.46

Tyrb (Y) 9.00 4.4 0.49 0.49 0.49 9.17 9.00 0.00

Met (M) 10.00 3.23 0.32 0.32 FALSE 6.73 7.00 30.00

Val (V) 35.00 11.6 0.33 0.33 FALSE 24.17 24.00 31.43

Ile (I) 20.00 6.85 0.34 0.34 FALSE 14.27 14.00 30.00

Leu (L) 18.00 9.81 0.55 0.55 0.55 20.44 20.00 11.11

Phe (F) 14.00 6.62 0.47 0.47 0.47 13.79 14.00 0.00




PMOL PROTEIN 0.48




Table 2.6S (3) Amino acid composition of trimer of modified GAPDH.

198

NAME/ID# GAPDH(Rabbit)

Tetramer Injection Id: 1499





His (H) 11.00 3.68 0.33 0.33 0.33 12.32 12.00 9.09

Asn (N) 17.00 0.00 FALSE

Ser (S) 19.00 10.35 0.54 FALSE FALSE 34.64 35.00 84.21

Gln (Q) 6.00 0.00 FALSE

Arg (R) 10.00 3.76 0.38 FALSE FALSE 12.59 13.00 30.00

Gly (G) 32.00 16.2 0.51 FALSE FALSE 54.22 54.00 68.75

Asp (D) 20.00 6.64 0.33 0.33 0.33 22.23 22.00 10.00

Glu (E) 13.00 3.54 0.27 0.27 0.27 11.85 12.00 7.69

Thr (T) 21.00 4.94 0.24 0.24 FALSE 16.53 17.00 19.05

Ala (A) 34.00 7.87 0.23 0.23 FALSE 26.34 26.00 23.53

Pro (P) 11.00 3.33 0.30 0.30 0.30 11.15 11.00 0.00

Lys (K) 26.00 4.21 0.16 0.16 FALSE 14.09 14.00 46.15

Tyrb (Y) 9.00 2.27 0.25 0.25 0.25 7.60 8.00 11.11

Met (M) 10.00 1.6 0.16 0.16 FALSE 5.36 5.00 50.00

Val (V) 35.00 6.7 0.19 0.19 FALSE 22.43 22.00 37.14

Ile (I) 20.00 4.05 0.20 0.20 FALSE 13.56 14.00 30.00

Leu (L) 18.00 6.14 0.34 FALSE FALSE 20.55 21.00 16.67

Phe (F) 14.00 3.49 0.25 0.25 FALSE 11.68 12.00 14.29




PMOL PROTEIN 0.29




Table 2.6S (4) Amino acid composition of tetramer of modified GAPDH. Table 2.6S Amino acid analysis template and amino acid composition of dimer, trimer and tetramer of modified GAPDH.

199

Tryptophan Peptide

Iso4 Fluorescence Tag

TIC Mass

Figure 2.9S Solution trypsin digested unmodified GAPDH was monitored by iso4 fluorescence tag and tryptophan peptides fluorescence channels.

Tryptophan Peptide

Iso4 Fluorescence Tag

TIC Mass

Figure 2.10S Solution trypsin digested fluorescently labeled iso[4]LGE2 modified GAPDH was monitored by iso4 fluorescence tag and tryptophan peptides fluorescence channels.

200

Sequence Molecular weight Peptides

270-321 5787.6488 GILGYTEDQVVSCDFNSATHSSTFDAGAGIALNDHFVKLISWYDNEFGYSNR 262-307 4853.2878 QASEGPLKGILGYTEDQVVSCDFNSATHSSTFDAGAGIALNDHFVK 144-184 4434.2501 IVSNASCTTNCLAPLAKVIHDHFGIVEGLMTTVHAITATQ K 19-53 4023.8811 AAFNSGKVDVVAINDPFIDLHYMVYMFQYDSTHGK 26-59 4017.9069 VDVVAINDPFIDLHYMVYMFQYDSTHGKFHGTVK 161-192 3374.7292 VIHDHFGIVEGLMTTVHAITATQKTVDGPSGK 117-143 2964.4361 VIISAPSADAPMFVMGVNHEKYDNSLK 79-105 2930.3643 DPANIKWGDAGAEYVVESTGVFTTMEK 85-111 2869.3592 WGDAGAEYVVESTGVFTTMEKAGAHLK 138-160 2539.241 YDNSLKIVSNASCTTNCLAPLAK 199-217 1723.958 GAAQNIIPASTGAAKAVGK 218-232 1660.897 VIPELNGKLTGMAFR 65-78 1600.93 LVINGKAITIFQER 322-333 1389.6632 VVDLMVHMASKE 214-225 1223.7237 AVGKVIPELNGK 185-195 1214.6407 TVDGPSGKLWR 54-64 1186.6094 FHGTVKAENGK 1-11 1178.623 MVKVGVNGFGR 259-269 1154.6659 VVKQASEGPLK 60-70 1141.6455 AENGKLVINGK 250-257 922.476 AAKYDDIK 106-115 908.5192 AGAHLKGGAK 253-258 780.4017 YDDIKK 247-252 658.4013 LEKAAK 112-116 487.2867 GGAKR 258-261 472.3373 KVVK

Table 2.7S Calculated tryptic digested GAPDH peptides with one miss cleavage. All cysteines have been treated with iodoacetamide to form carbamidomethyl-cysteine. Methionines have been oxidized to form methionine sulfoxide. Using monoisotopic masses of the occurring amino acid residues and giving peptide masses as [M].

201

Table 2.8S Mass of cross-linked peptides [M], including +1 to +4 charges

203

TOF MS ES+ 282

m/z

m/z

TOF MSMS 640.97 ES+

32

Figure 2.11S TOF MS and MSMS of ion 640.9626.

171

Table 3.1S Molecular weight summary of Acetyl-Gly-Lys-O-Methyl Ester/Amyloid (11-17) “EVHHQKL” peptide reactions with iso[4]LGE2/iso[4]LGE2-Fluo tag

(1) Acetyl-Gly-Lys-O-Methyl Ester reaction with iso[4]LGE2 Acetyl-Gly-Lys-O-Methyl Ester (Average molecular weight): 259.3056 H:1.008 H2O:18.0153 Adducts-- no fluorescence tag

Adduct Formula

Adduct MW

Product MW

Plus one charge (+1)

Plus two charges (+2)

Pyrrole C20H28O3 316.4405 575.7461 576.7541 288.88105 Pyrrole -H2O C20H26O2 298.4252 557.7308 558.7388 279.8734 Lactam C20H28O4 332.4399 591.7455 592.7535 296.88075 Lactam-H2O C20H26O3 314.4246 573.7302 574.7382 287.8731 Hydroxylactam C20H28O5 348.4393 607.7449 608.7529 304.88045 Hydroxylactam-H2O C20H26O4 330.4240 589.7296 590.7376 295.8728 Hydroxylactam-2H2O C20H24O3 312.4088 571.7144 572.7224 286.8652

Cross-link C20H33O4 337.4796 853.0668 854.0748 427.5414 Cross-link-H2O C20H31O3 319.4643 835.0515 836.0595 418.53375 Cross-link-2H2O C20H29O2 301.4491 817.0362 818.0442 409.5261 Bis-pyrrole C40H58O6 634.8969 1149.4761 1150.4841 575.74605 Bis-pyrrole-H2O C40H56O5 616.8816 1131.4608 1132.4688 566.7384 Bis-pyrrole-2H2O C40H54O4 598.8664 1113.4455 1114.4535 557.73075 Tris-pyrrole C60H88O9 953.3353 1725.2041 1726.2121 863.6101 Tris-pyrrole-H2O C60H86O8 935.3200 1707.1888 1708.1968 854.6024 Tris-pyrrole-2H2O C60H84O7 917.3047 1689.1735 1690.1815 845.5948 Tris-pyrrole-3H2O C60H82O6 899.2894 1671.1582 1672.1662 836.5871 Acetyl-Gly-Lys-O-Methyl Ester (Monoisotopic molecular weight): 259.1532 H:1.0078 H2O:18.0106 Adducts-- no fluorescence tag

Adduct Formula

Adduct MW

Product MW



Pyrrole C20H28O3 316.2038 575.3570 576.3648 288.6863 Pyrrole -H2O C20H26O2 298.1933 557.3465 558.3543 279.68105 Lactam C20H28O4 332.1988 591.3520 592.3598 296.6838 Lactam-H2O C20H26O3 314.1882 573.3414 574.3492 287.6785 Hydroxylactam C20H28O5 348.1937 607.3469 608.3547 304.68125 Hydroxylactam-H2O C20H26O4 330.1831 589.3363 590.3441 295.67595 Hydroxylactam-2H2O C20H24O3 312.1725 571.3257 572.3335 286.67065 Cross-link C20H33O4 337.2379 852.5209 853.5287 427.26825 Cross-link-H2O C20H31O3 319.2273 834.5103 835.5181 418.26295 Cross-link-2H2O C20H29O2 301.2168 816.4997 817.5075 409.25765 Bis-pyrrole C40H58O6 634.4233 1148.6985 1149.7063 575.35705 Bis-pyrrole-H2O C40H56O5 616.4128 1130.6879 1131.6957 566.35175 Bis-pyrrole-2H2O C40H54O4 598.4022 1112.6773 1113.6851 557.34645 Tris-pyrrole C60H88O9 952.6428 1724.0556 1725.0634 863.0356 Tris-pyrrole-H2O C60H86O8 934.6322 1706.0450 1707.0528 854.0303 Tris-pyrrole-2H2O C60H84O7 916.6216 1688.0344 1689.0422 845.0250 Tris-pyrrole-3H2O C60H82O6 898.6110 1670.0238 1671.0316 836.0197

172

(2) Acetyl-Gly-Lys-O-Methyl Ester reaction with iso[4]LGE2-Fluo tag Acetyl-Gly-Lys-O-Methyl Ester (Monoisotopic molecular weight): 259.1532 H:1.0078 H2O:18.0106 Adducts-- no fluorescence tag

Adduct Formula

Adduct MW

Product MW



Pyrrole C30H41N5O5S 583.2828 842.4360 843.4438 422.2258 Pyrrole -H2O C30H39N5O4S 565.2723 824.4255 825.4333 413.22055 Lactam C30H41N5O6S 599,2778 858.4310 859.4388 430.2233 Lactam-H2O C30H39N5O5S 581.2672 840.4204 841.4282 421.2180 Hydroxylactam C30H41N5O7S 615.2727 874.4259 875.4337 438.22075 Hydroxylactam-H2O C30H39N5O6S 597.2621 856.4153 857.4231 429.21545 Hydroxylactam-2H2O C30H37N5O5S 579.2515 838.4047 839.4125 420.21015 Cross-link C30H46N5O6S 604.3168 1119.5999 1120.6077 560.80775 Cross-link-H2O C30H44N5O5S 586.3062 1101.5893 1102.5971 551.80245 Cross-link-2H2O C30H42N5O4S 568.2956 1083.5787 1084.5865 542.79715 Bis-pyrrole C60H84N10O10S2 1168.5813 1682.8562 1683.8640 842.4359 Bis-pyrrole-H2O C60H82N10O9S2 1150.5707 1664.8456 1665.8534 833.4306 Bis-pyrrole-2H2O C60H80N10O8S2 1132.5601 1646.835 1647.8428 824.4253

(3) Amyloid (11-17) “EVHHQKL” peptide reaction with iso[4]LGE2 EVHHQKL (Average molecular weight): 889.99854, (+1) 891.00593, (+2) 446.0666 H:1.008 H2O:18.0153 Adducts-- no fluorescence tag

Adduct Formula

Adduct MW

Product MW



Plus three charges (+3)

Plus four charges (+4)

Pyrrole C20H28O3 316.4405 1206.4390 1207.4470 604.2275 403.15433 302.61775 Pyrrole -H2O C20H26O2 298.4252 1188.4237 1189.4317 595.21985 397.14923 298.11392 Lactam C20H28O4 332.4399 1222.4384 1223.4464 612.2272 408.48746 306.6176 Lactam-H2O C20H26O3 314.4246 1204.4231 1205.4311 603.21955 402.48236 302.11377 Hydroxylactam C20H28O5 348.4393 1238.4378 1239.4458 620.2269 413.8206 310.61745 Hydroxylactam-H2O C20H26O4 330.4240 1220.4225 1221.4305 611.21925 407.8155 306.11362 Hydroxylactam-2H2O

C20H24O3 312.4088 1202.4073 1203.4153 602.21165 401.81043 301.60982

Cross-link C20H33O4 337.4796 2114.4526 2115.4606 1058.2343 705.82553 529.62115 Cross-link-H2O C20H31O3 319.4643 2096.4373 2097.4453 1049.2266 699.82043 525.11732 Cross-link-2H2O C20H29O2 301.4491 2078.4221 2079.4301 1040.2190 693.81536 520.61352 Bis-pyrrole C40H58O6 634.8969 2410.8619 2411.8699 1206.4389 804.62863 603.72347 Bis-pyrrole-H2O C40H56O5 616.8816 2392.8466 2393.8546 1197.4313 798.62353 599.21965 Bis-pyrrole-2H2O C40H54O4 598.8664 2374.8314 2375.8394 1188.4237 792.61846 594.71585 Tris-pyrrole C60H88O9 953.3353 3617.2829 3618.2909 1809.6495 1206.7690 905.3287 Tris-pyrrole-H2O C60H86O8 935.3200 3599.2676 3600.2756 1800.6418 1200.7639 900.8249 Tris-pyrrole-2H2O C60H84O7 917.3047 3581.2523 3582.2603 1791.6342 1194.7588 896.3211 Tris-pyrrole-3H2O C60H82O6 899.2894 3563.2370 3564.2450 1782.6265 1188.7537 891.8173 Pyrrole+Lactam C40H58O7 650.8963 2426.8613 2427.8693 1214.4387 809.9618 607.7233 Pyr+Lac-H2O C40H56O6 632.8810 2408.8460 2409.8540 1205.4310 803.9567 603.2195 Pyr+Lac-2H2O C40H54O5 614.8658 2390.8307 2391.8387 1196.4234 797.9516 598.7157

173

EVHHQKL (Monoisotopic molecular weight): 889.476977, (+1) 890.454253, (+2) 445.745765 H:1.0078 H2O:18.0106 Adducts-- no fluorescence tag

Adduct Formula

Adduct MW

Product MW





Pyrrole C20H28O3 316.2038 1205.6808 1206.6886 603.8482 402.9014 302.4280 Pyrrole -H2O C20H26O2 298.1933 1187.6703 1188.6781 594.84295 396.8979 297.9254 Lactam C20H28O4 332.1988 1221.6750 1222.6828 611.8453 408.2328 306.4266 Lactam-H2O C20H26O3 314.1882 1203.6652 1204.6730 602.8404 402.2295 301.9241 Hydroxylactam C20H28O5 348.1937 1237.6707 1238.6785 619.84315 413.5647 310.4255 Hydroxylactam-H2O C20H26O4 330.1831 1219.6601 1220.6679 610.83785 407.5612 305.9228 Hydroxylactam-2H2O

C20H24O3 312.1725 1201.6495 1202.6573 601.83255 401.5576 301.4202

Cross-link C20H33O4 337.2379 2113.1685 2114.1763 1057.5921 705.3973 529.2999 Cross-link-H2O C20H31O3 319.2273 2095.1579 2096.1657 1048.5867 699.3938 524.7973 Cross-link-2H2O C20H29O2 301.2168 2077.1473 2078.1551 1039.5814 693.39023 520.2946 Bis-pyrrole C40H58O6 634.4233 2409.3461 2410.3539 1205.6808 804.12316 603.3443 Bis-pyrrole-H2O C40H56O5 616.4128 2391.3355 2392.3433 1196.6755 798.11963 598.8417 Bis-pyrrole-2H2O C40H54O4 598.4022 2373.3249 2374.3327 1187.6702 792.1161 594.3390 Tris-pyrrole C60H88O9 952.6428 3617.9588 3618.9666 1809.9872 1206.9941 905.4975 Tris-pyrrole-H2O C60H86O8 934.6322 3599.9482 3600.9560 1800.9819 1200.9905 900.9948 Tris-pyrrole-2H2O C60H84O7 916.6216 3581.9376 3582.9454 1791.9766 1194.9870 896.4922 Tris-pyrrole-3H2O C60H82O6 898.6110 3563.9270 3564.9348 1782.9713 1188.9835 891.9895 Pyrrole+Lactam C40H58O7 650.4183 2425.3411 2426.3489 1213.6783 809.4548 607.3431 Pyr+Lac-H2O C40H56O6 632.4077 2407.3305 2408.3383 1204.6730 803.4513 602.8404 Pyr+Lac-2H2O C40H54O5 614.3971 2389.3209 2390.3287 1195.6682 797.4481 598.3380

(4) Amyloid (11-17) “EVHHQKL” peptide reaction with iso[4]LGE2-Fluo tag EVHHQKL (Monoisotopic molecular weight): 889.476977, (+1) 890.454253, (+2) 445.745765 H:1.0078 H2O:18.0106

Adducts-- no fluorescence tag

Adduct Formula

Adduct MW

Product MW





Pyrrole C30H41N5O5S 583.2828 1472.7598 1473.7676 737.3877 491.9277 369.19775 Pyrrole -H2O C30H39N5O4S 565.2723 1454.7493 1455.7571 728.38245 485.9242 364.69513 Lactam C30H41N5O6S 599,2778 1488.7548 1489.7626 745.3852 497.2594 373.1965 Lactam-H2O C30H39N5O5S 581.2672 1470.7442 1471.7520 736.3799 491.2559 368.69385 Hydroxylactam C30H41N5O7S 615.2727 1504.7497 1505.7575 753.38265 502.5910 377.19523 Hydroxylactam-H2O C30H39N5O6S 597.2621 1486.7391 1487.7469 744.37735 496.5875 372.69258 Hydroxylactam-2H2O

C30H37N5O5S 579.2515 1468.7285 1469.7363 735.37502 490.5840 368.1899

Cross-link C30H46N5O6S 604.3168 3266.6997 2381.2553 1191.1315 1089.9077 817.6827 Cross-link-H2O C30H44N5O5S 586.3062 3248.6891 2363.2447 1182.1262 1083.9042 813.1801 Cross-link-2H2O C30H42N5O4S 568.2956 3230.6785 2345.2341 1173.1209 1077.9006 808.6774 Bis-pyrrole C60H84N10O10S2 1168.5813 3830.9642 2944.5119 1472.7598 1277.9959 958.7489 Bis-pyrrole-H2O C60H82N10O9S2 1150.5707 3812.9536 2926.5013 1463.7545 1271.9923 954.2462 Bis-pyrrole-2H2O C60H80N10O8S2 1132.5601 3794.9430 2908.4907 1454.7492 1265.9888 949.7436

174

Figure 3.1S Acetyl-Gly-Lys-O-Methyl ester reactions with iso[4]LGE2 in different molar ratio and time point

(1) Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (Dipeptide: Iso4 = 2000:1 molar ratio) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 8 days, 15 days, 21 days and 50 days.

Modified One Adducts

Crosslink

Bispyrrole Trispyrrole

Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 1 day

Trispyrrole

-H2O-2H2O

-3H2O

A

Crosslink


BispyrroleTrispyrrole

Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 4 days

B

175

Trispyrrole

Trispyrrole

Bispyrrole

Crosslink

Modified One Adducts Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (2000:1) in

50 mM NEM OAc pH 8.6 for 8 days-H2O

-2H2O

-3H2O

C



Crosslink


Bispyrrole

-H2O

-2H2O

+16

+16+16

+16

D

176

TrispyrroleBispyrrole

Crosslink

Modified One Adducts Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (2000:1) in

50 mM NEM OAc pH 8.6 for 21 days

Bispyrrole

-H2O

-2H2O

+16+16

+16

+16

E

TrispyrroleBispyrroleCrosslink



Trispyrrole-2H2O -H2O

-3H2O

+16

+16

+16

F

177



Crosslink

Modified One Adducts Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (1000:1)

in 50 mM NEM OAc pH 8.6 for 1 day

Trispyrrole-H2O-2H2O-3H2O

A

Crosslink




B

178

Crosslink


Trispyrrole-2H2O-H2O

-3H2O

Modified One Adducts Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (1000:1) in 50 mM

NEM OAc pH 8.6 for 8 daysC


Crosslink

Modified One Adducts Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (1000:1) in 50 mM

NEM OAc pH 8.6 for 15 daysBispyrrole

-H2O

-2H2O

+16

+16+16

+16

D

179


Crosslink



Bispyrrole

-H2O

-2H2O

+16+16

+16

+16

+16

E

Bispyrrole

Trispyrrole

Crosslink



Trispyrrole-H2O-2H2O

-3H2O+16

+16

+16

F

180


Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 1 day


Crosslink


Trispyrrole-H2O-2H2O

-3H2O

+16 +16

A


Crosslink


Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 4 days Bispyrrole

-H2O

-2H2O

+16

+16

+16

+16

B

181

Trispyrrole

Trispyrrole

Bispyrrole

Crosslink


Acetyl-Gly-Lys-O-Methyl ester reacts with Iso[4] no fluorescence tag (200:1) in 50 mM NEM OAc pH 8.6 for 8 days

-H2O

-2H2O

-3H2O

+16

+16

+16

+16

C

Trispyrrole

Bispyrrole

Crosslink



Bispyrrole

-2H2O

-H2O

+16

+16

+16

+16

D

182


Crosslink



Bispyrrole

-H2O

-2H2O

+16

+16

+16

+16

+16

E



Crosslink


Trispyrrole

+16

+16

+16

-H2O-2H2O

-3H2O

F

183

Crosslink

-H2O

-2H2O

Pyrrole-H2O

Lactam-H2O

HydroxyLactam-H2O

-2H2O

A B

C D

Figure 3.2S ESI-MSMS of molecular ions at A) aminal cross-link, B) pyrrole, C) lactam and D) hydroxylactam corresponding to a iso[4]LGE2–(N-acetyl-Gly-Lys-OMe) adduct.

184

NH

O HN COOCH3

NH2O

+C

O

C5H11

OO

OH

Crosslink N C

NH

C5H11

OHO

OH

P

P

-H2O

MW 853.5(+1)

MW 835.5(+1)

MW 558.4(+1)Like Pyrrole-H2O

MW 436.3(+1)

Minus MW 122

N C OHO

P C5H11+

MS/MS

MS/MSMW 853.5(+1)835.5(+1)817.5(+1)

(-H2O)(-2H2O)

MW 473.3(+1)HN C

NH

C5H11

OHO

OHP

N C

NH

C5H11

OHO

P

P

N CC5H11

O

P

N C

NH

C5H11

OH

OHO

OH

P

P

OH OH

OH OH

Pyrrole MW 576.4(+1)-H2O MW 558.4(+1)

Lactam MW 592.4(+1)N C

C5H11

OHO

P MW 475.4(+1)Minus MW 99-H2O MW 574.4(+1)

O

MW 415.4(+1)Minus MW 60

HydroxyLactam 608.4(+1)N C

C5H11

OHO

P

O-2H2O MW 572.4(+1)-H2O MW 590.4(+1)

NH

OHN COOCH3

NH2O

+ C

O

C5H11

OO

OH

MW 436.3(+1)Minus MW 122

N C OHO

PC5H11+

N CC5H11

OHO

P

OH

MW 473.4(+1)Minus MW 99 MW 413.4(+1)Minus MW 60

One Site Modification

Scheme 3.1S Possible fragmentation of (N-acetyl-Gly-Lys-OMe)-iso[4]LGE2 adducts.

185

Figure 3.3S Acetyl-Gly-Lys-O-Methyl ester reactions with iso[4]LGE2-Fluo tag in different molar ratio and time point

(1) Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag (Dipeptide : Iso4 = 2000:1) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 15 days, 21 days and 50 days.

Bispyrrole

Crosslink

Modified One Adducts Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag

(2000:1) in 50 mM NEM OAc pH 8.6 for 1 day

Bispyrrole

-H2O-2H2O

A

Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag (2000:1) in 50 mM NEM OAc pH 8.6 for 4 days


Bispyrrole

Crosslink

B

186

Bispyrrole

Crosslink


Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag (2000:1) in 50 mM NEM OAc pH 8.6 for 15 days Crosslink

-H2O

-2H2O

C

Crosslink


Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag (2000:1) in 50 mM NEMOAc pH 8.6 for 21 days

Crosslink

-H2O

-2H2O

D

187


Crosslink

Crosslink

-H2O

-2H2O


E

(2) Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag (Dipeptide : Iso4 = 200:1) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 15 days, 21 days and 50 days.

Crosslink


Bispyrrole

Bispyrrole-H2O-2H2O

Acetyl-Gly-Lys-O-Methyl ester reacts with iso[4]LGE2-Fluo tag (200:1) in 50 mM NEMOAc pH 8.6 for 1 day

A

188


Bispyrrole

Crosslink


B



Crosslink

Crosslink

-H2O

-2H2O

C

189

Bispyrrole

Crosslink



Crosslink

-2H2O

-H2O

D

Crosslink

Modified One Adducts Crosslink


-H2O

-2H2O

E

190

HydroxyLactam-2H2O

HydroxyLactam-H2O

HydroxyLactam

Lactam-H2O

Lactam

Pyrrole-H2O

Pyrrole

Crosslink

-H2O

-2H2O

A B

C D

Figure 3.4S: ESI-MSMS of molecular ions at A) animal cross-link, B) pyrrole, C) lactam and D) hydroxylactam corresponding to a Iso[4]LGE2-FluoTag - (N-acetyl-Gly-Lys-OMe) adduct.

191

NH

O HN COOCH3

NH2O

+ C

O

C5H11

O

NHCH2CH2NHO

NO

N

SO2N

OH

CrosslinkN C

NH

C5H11

OH

FluoTagO

OH

P

P

-H2OMW 1120.6(+1) MW 1102.6(+1)

MW 825.5(+1)Like Pyrrole-H2O 703.4(+1)

-MW 122 N C FluoTagO

P

C5H11+

MS/MS

N C

NH

C5H11

OH

FluoTagO

P

P

N CC5H11

FluoTagO

P

MS/MSMW 1120.6(+1)

1102.6(+1)

1084.6(+1)

(-H2O)

(-2H2O)

N C

NH

C5H11

OH

FluoTag

O

OH

P

P

740.5(+1)HN C

NH

C5H11

FluoTag

O

OHP

Pyrrole MW 843.4(+1)

Pyrrole-H2O MW 825.4(+1)

NH

O HN COOCH3

NH2O

+ C

O

C5H11

O

NHCH2CH2NHO

NO

N

SO2N

OH

N CC5H11

FluoTagO

P

Lactam MW 859.4(+1) N CC5H11

FluoTagO

P 742.4(+1)-MW 99

Lactam-H2O MW 841.4(+1)O

682.4(+1) -MW 60

HydroxyLactam 875.4(+1) N CC5H11

FluoTagO

P

OHydroxyLactam-2H2O 839.4(+1)HydroxyLactam-H2O 857.4(+1) 740.4(+1)

-MW 99680.4(+1)

-MW 60

703.4(+1)-MW 122 N C FluoTag

O

P

C5H11+

Scheme 3.2S Possible fragmentation of (N-acetyl-Gly-Lys-OMe)-iso[4]LGE2-FluoTag adducts.

192

Figure 3.5S Amyloid (11-17) “EVHHQKL” peptide reactions with iso[4]LGE2 in different molar ratio and time point

(1) Amyloid (11-17) “EVHHQKL” reacts with iso[4]LGE2 (Peptide: Iso4 = 2000:1) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 15 days, 21 days and 50 days.


Crosslink Bispyrrole Trispyrrole

Bispyrrole

-2H2O-H2O

Amyloid (11-17) "EVHHQKL" reacts with iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 1 day

+16

A


Amyloid (11-17) "EVHHQKL" reacts with iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 4 days


Bispyrrole

-H2O

-2H2O+16

B

193

Bispyrrole

Bispyrrole

TrispyrroleCrosslink

Amyloid (11-17) "EVHHQKL" reacts with iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 15 days

-H2O

-2H2O+16

+16

Modified One Adducts C

Bispyrrole TrispyrroleCrosslink

Modified One Adducts Amyloid (11-17) "EVHHQKL" reacts with

iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 21 days

Bispyrrole

-H2O

-2H2O

+16

+16

+16

D

194

Crosslink

Bispyrrole



Amyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2 (2000:1) in 50 mM NEM OAc pH 8.6 for 50 days

-H2O

-2H2O

+16

+16

+16+16

E

(2) Amyloid (11-17) “EVHHQKL” reacts with iso[4]LGE2 (Peptide: Iso4 = 200:1) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 7 days, 15 days, 21 days and 50 days.



Amyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 1 day

Bispyrrole

-H2O

-2H2O

+16

+16

A

195

TrispyrroleBispyrroleCrosslink

Modified One Adducts Amyloid (11-17) "EVHHQKL" reacts

with iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 4 days

Bispyrrole

-H2O

-2H2O

+16

B

-2H2OBispyrrole

+16

-H2OAmyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 7 days


Bispyrrole TrispyrroleCrosslink

C

196


Bispyrrole

-H2O

-2H2O +16


with iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 15 days

Bispyrrole

D



with iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 21days

Bispyrrole

-2H2O

-H2O

+16

+16

E

197

-2H2O

-H2O

Bispyrrole

+16

+16

+16

Amyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2 (200:1) in 50 mM NEM OAc pH 8.6 for 50 days



F

Figure 3.6S Amyloid (11-17) “EVHHQKL” peptide reactions with iso[4]LGE2-Fluo tag in different molar ratio and time point

(1) Amyloid (11-17) “EVHHQKL” reacts with iso[4]LGE2-Fluo tag (Peptide: Iso4 = 2000:1) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 7 days, 15 days, 21 days and 50 days.

Amyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2-FluoTag (2000:1)in 50 mM NEM OAc pH 8.6 for 1 day


No Obvious crosslink or bispyrrole peak formed

A

198


Amyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2-FluoTag (2000:1)in 50 mM NEM OAc pH 8.6 for 4 days

Crosslink

Crosslink-H2OB

Crosslink

Crosslink

-H2O



C

199

Crosslink



Crosslink

-H2O

-2H2O

D

Crosslink



Crosslink

-H2O

E

200



Crosslink

Crosslink

-H2O

F

(2) Amyloid (11-17) “EVHHQKL” reacts with iso[4]LGE2-Fluo tag (Peptide: Iso4 = 200:1) in 50 mM NEM OAc pH 8.6 for 1 day, 4 days, 7 days, 15 days, 21 days and 50 days.


No Obvious crosslink or bispyrrole peak formed

Amyloid (11-17) "EVHHQKL" reactswith iso[4]LGE2-FluoTag (200:1)in 50 mM NEM OAc pH 8.6 for 1 day

A

201


Crosslink

Crosslink


B

Crosslink

-H2O

Crosslink



C

202

Crosslink

Crosslink

-H2O



D

Crosslink-H2O


Crosslink


E

203



Crosslink

-H2O

Crosslink

F

UPLC purified pyrrole

No oxidized crosslink and bispyrrole

Pyrrole-H2O

NO Acetyl-Gly-Lys-O-Methyl ester and Iso[4]

Pyrrole-H2O

Figure 4.1S MALDI-TOF spectrums of iso[4]LGE2-pyrrole

204

Iso[4]LGE2 purified with C18 toptip Acetyl-Gly-Lys-O-Methyl ester

Figure 4.2S MALDI-TOF spectrums of iso[4]LGE2 and Acetyl-Gly-Lys-O-methyl ester ---- indicating new peaks besides the matrix.

No oxidized crosslink and bispyrrole

NO GK didpeptide and Iso[4]UPLC purified pyrrolePyrrole-H2O Pyrrole-H2O After 2 days reaction

under Air

No crosslink formed, a little bispyrrole formed

Pyrrole-H2O After 6 days reaction under Air

No crosslink formed, some bispyrrole formed

More than 8 days incubationPyrrole-H2O

Bispyrrole peaks

Lactam

Hydroxylactam

Figure 4.3S MALDI-TOF spectrums of HPLC-purified iso[4]LGE2-pyrrole reaction under air within 8 days.

205

Pyrrole-H2OPure Iso[4]LGE2-pyrrole was added Ac-Lysineand reacted for 2 days.Iso[4]LGE2-pyrrole didn't oxidize through this way.It didn't relate with molar ratio and pH.

No oxidized products were formed

Figure 4.4S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with Nε-acetyl-L-lysine under air for 2 days.

Pyrrole-H2O Pure Iso[4]LGE2-pyrrole was addedL-Lysine (1:10 mlolar ratio),reacted under air for 2 days.

No oxidized products were formed

Figure 4.5S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with L-

lysine under air for 2 days.

206

Pyrrole-H2O

Amyloid peptide

Pure pyrrole was added Amyloid peptide in H2O (1:10 mlolar ratio), reacted under air for 6 h.No other oxidized peak formedNo Lactam or Hydrolactam formed

Pyrrole-H2OAmyloid peptide Pyrrole-H2O

Amyloid peptide

Pure pyrrole was added Amyloid peptide in H2O (1:10 mlolar ratio), reacted under air overnight.No other oxidized peak formedNo Lactam or Hydrolactam formed

Pure pyrrole was added Amyloid peptide in H2O (1:10 mlolar ratio), reacted under air for 2 days.No other oxidized peak formedNo Lactam or Hydrolactam formed

Figure 4.6S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with β-Amyloid peptide under air for 2 days.

Pyrrole-H2O

The peak 544 was shown up after adding Ethanolamine.

Pure pyrrole was added ethanolamine equal ratio and reacted for 2 days.Pyrrole decreased through this way.pH=7.

Pure pyrrole was added ethanolamine much more excess and reacted for 2 h.Pyrrole couldn't be observed.pH=7.

The peak 544 was shown up after adding Ethanolamine.

No pyrrole-H2O

Figure 4.7S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with ethanolamine under air.

207

Pure pyrrole was added GK Dipeptide----Reacted 1h under Air

Pyrrole-H2O

Pure pyrrole was added GK Dipeptide----Reacted 8h under Air

Hydroxylactam-2H2O

Pyrrole-H2O

Hydroxylactam-2H2OPure pyrrole was added GK Dipeptide----Reacted 1 day under Air

Pyrrole-H2O

Pyrrole-H2O

Hydroxylactam-2H2O Pure pyrrole was added GK Dipeptide----Reacted 2 days under Air

GK dipeptide in H2O GK dipeptide in H2O

GK dipeptide in H2OGK dipeptide in H2O

(From BACHEM) (From BACHEM)

(From BACHEM)(From BACHEM)

Hydroxylactam-2H2O

Figure 4.8S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with Acetyl-Gly-Lys-O-Methyl Ester under air.

Pyrrole-H2O Pyrrole-H2O

Pure pyrrole was addedUPLC purified GK dipeptide--reacted under air for 2 h.

Pure pyrrole was addedUPLC purified GK dipeptide--reacted under air for 6 h.

No other oxidized product formed No other oxidized product formedNo Lactam or Hydroxylactam formed No Lactam or Hydroxylactam formed

Pyrrole-H2O Pure pyrrole was addedUPLC purified GK dipeptide--reacted under air for 2 days.

No Lactam or Hydroxylactam formedNo other oxidized product formed

Pyrrole-H2O Pure pyrrole was addedUPLC purified GK dipeptide--reacted under air for 6 days.

No Lactam or Hydroxylactam formedNo other oxidized product formed

Formed Bispyrrole

Figure 4.9S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with HPLC-purified Acetyl-Gly-Lys-O-Methyl Ester under air.

208

Table 4.1S Summaries of HPLC-purified iso[4]LGE2-pyrrole reaction with different chemicals.

(a) HPLC-purified iso[4]LGE2-pyrrole reactions with Acetyl-Gly-Lys-O-Methyl Ester and Initiator (2,2'-Azobis(2-methylpropionamidine) dihydrochloride)

Molar ratio 2 h reaction Overnight reaction A week reaction Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =1:0.1

Pyrrole—no changes Pyrrole—decreased little bit, very little oxidized product formed

Almost no pyrrole, little oxidized product formed

Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =1:1

Pyrrole—decreased little bit, little oxidized product formed

Pyrrole—decreased little bit, more oxidized product formed

Almost no pyrrole, much oxidized products formed

Pyrrole: Initiator=1:0.1 Pyrrole—decreased, no oxidized product formed

No pyrrole or oxidized product


Pyrrole: Initiator=1:1 No pyrrole or oxidized product



Molar ratio 2 h reaction Overnight reaction 2 days reaction Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =1:10

Pyrrole—decreased little bit, much oxidized product formed


Pyrrole—decreased, much more oxidized products formed

Pyrrole: Initiator=1:0.01 Pyrrole—no change, no oxidized product formed

Pyrrole— decreased, no oxidized product formed

Pyrrole— decreased, no oxidized product formed

Molar ratio 4 h reaction Overnight reaction Pyrrole: Initiator=1:0.05 Very little pyrrole left and no oxidation No pyrrole left and no oxidation

(b) HPLC-purified iso[4]LGE2-pyrrole reactions with Vitamin C, Acetyl-Gly-Lys-O-Methyl Ester and Initiator (2,2'-Azobis(2-methylpropionamidine) dihydrochloride)

Molar ratio 2 h reaction Overnight reaction A week reaction Vitamin C: Pyrrole=0.1:1 Pyrrole—no changes

No oxidized product formed No pyrrole No oxidized product formed

No pyrrole and formed a peak (+670), no other oxidized products detected

Vitamin C: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =0.1:1:0.1

Pyrrole—no changes No oxidized product formed

Pyrrole—decreased little bit, very little oxidized product formed

Pyrrole—decreased little bit, very little oxidized product formed. Also formed peak (+670)

Vitamin C: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =0.1:1:1



Pyrrole—decreased little bit, much more oxidized product formed. Also formed peak (+670)

Vitamin C: Pyrrole: Initiator=0.1:1:0.1


No pyrrole No oxidized product formed

No pyrrole, no oxidized product formed. Also formed peak (+670)

Vitamin C: Pyrrole: Initiator=0.1:1:1


No pyrrole No oxidized product formed

No pyrrole, no oxidized product formed. Also formed peak (+670), and some other unknown peaks

Molar ratio 2 h reaction Overnight reaction 2 days reaction Vitamin C: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =0.01:1:10

Pyrrole—decreased little bit, much oxidized product formed

Pyrrole—decreased, more oxidized product formed

Pyrrole—decreased, much more oxidized products formed

Vitamin C: Pyrrole: Initiator=0.01:1:0.01

Pyrrole—no changes. No oxidized product formed

Pyrrole— decreased. No oxidized product formed

Pyrrole— decreased. No oxidized product formed

(c) HPLC-purified iso[4]LGE2-pyrrole reactions with Vitamin E, Acetyl-Gly-Lys-O-Methyl

Ester and Initiator (2,2'-Azobis(2-methylpropionamidine) dihydrochloride) Molar ratio 2 h reaction Overnight reaction A week reaction Vitamin E: Pyrrole=0.1:1 Pyrrole—no changes

No oxidized product formed No pyrrole or oxidized product


209

Vitamin E: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =0.1:1:0.1


No pyrrole, formed very little oxidized products

No pyrrole, some known peaks

Vitamin E: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =0.1:1:1


Pyrrole—decreased, little oxidized product formed


Vitamin E: Pyrrole: Initiator=0.1:1:0.1

Pyrrole—decreased, No oxidized product formed



Vitamin E: Pyrrole: Initiator=0.1:1:1

Pyrrole—decreased, No oxidized product formed



Molar ratio 2 h reaction Overnight reaction 2 days reaction Pyrrrole: Vitamin E=1: 0.01 Pyrrole—no changes. No

oxidized product formed Pyrrole— decreased. No oxidized product formed

No pyrrole left, no oxidized products and some unknown peaks

Molar ratio 4 h reaction Overnight reaction Pyrrole: Vitamin E:initiator =1: 0.01:0.01

No pyrrole left and no oxidation Pyrrole— decreased and no oxidation

(d) HPLC-purified iso[4]LGE2-pyrrole reactions with H atom donor ((R)-(-)-2-(2,5-

Dihydrophenyl)glycine), Acetyl-Gly-Lys-O-Methyl Ester and Initiator (2,2'-Azobis(2-methylpropionamidine) dihydrochloride)

Molar ratio 2 h reaction Overnight reaction A week reaction H atom Donor: Pyrrole=5:1 No pyrrole or oxidized

product No pyrrole or oxidized product


H atom Donor: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =5:1:0.1




H atom Donor: Pyrrole: Acetyl-Gly-Lys-O-Methyl Ester =5:1:1

No pyrrole, little oxidized products formed

No pyrrole, little oxidized products formed


H atom Donor: Pyrrole: Iniitator=5:1:0.1




H atom Donor: Pyrrole: Iniitator=5:1:1




Molar ratio 2 h reaction Overnight reaction 2 days reaction Pyrrrole: H atom dondor =5:1




Molar ratio 4 h reaction Overnight reaction Pyrrole: H atom Donor =1:1 Very little pyrrole left and no oxidation No pyrrole left and no oxidation Pyrrole:H atom Donor: Acetyl-Gly-Lys-O-Methyl Ester =2:1:1

Pyrrole—decreased, some oxidized product formed similar like pyrrole with Acetyl-Gly-Lys-O-Methyl Ester

(e) HPLC-purified iso[4]LGE2-pyrrole reactions with UV, rose bengal, methylene blue,

Co2(CO)8, H2O2 K2CO3, dimethyldioirane, microwave, Di-tert-butyl peroxide, riboflavin (VB2), K3Fe(CN)6, FeCl3, K2S2O8.

Molar ratio 30 min 1 h reaction 2 h reaction Pyrrole: rose bengal=1:1, UV, air No pyrrole or oxidized

product No pyrrole or oxidized product


Pyrrole: methylene blue =1:1, UV, air




UV light and air No pyrrole or oxidized product



Condition 2 min 8 min UV light and air Pyrrole—decreased. No oxidized product Pyrrole—decreased. No oxidized product

210

formed formed Condition 10 seconds 30 seconds Microwave and air Pyrrole—decreased. No oxidized product

formed Pyrrole—decreased. No oxidized product formed

Condition 5 min 20 min UV light and air, riboflavin (VB2)

No pyrrole or oxidized product No pyrrole or oxidized product

Condition Results Pyrrole: Co2(CO)8=1:1, overnight reaction No pyrrole or oxidized product Pyrrole: H2O2/K2CO3=1:1, overnight reaction No pyrrole or oxidized product Pyrrole: dimethyldioirane =1:1, 0.5 h reaction No pyrrole or oxidized product Pyrrole: Di-tert-butyl peroxide =1:1, 2 h reaction No pyrrole or oxidized product Catalytic amount of K3Fe(CN)6, 1 h reaction No pyrrole or oxidized product Catalytic amount of FeCl3, 1 h reaction No pyrrole or oxidized product Catalytic amount of K2S2O8, 1 h reaction No pyrrole or oxidized product

Peaks for bispyrrole

Pyrrole-H2OLactam-H2O

Hydroxylactam-2H2O

Hydroxylactam-H2O

Lactam

Hydroxylactam

Matrix Pure pyrrole was added 1 mol% TEMED and 2 mol% APS for 1.5 h 25 oC under air

Figure 4.10S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with APS and TEMED at 25 ℃ under air.

211

Bispyrrole

Hydrolactam-2H2O


Hydrolactam-2H2O

Pure pyrrole was added 1 mol% TEMED and 1 mol% APS 37 degree for 1h

Lactam-H2O

Hydrolactam-H2OHydrolactam


Hydrolactam-2H2O

Hydrolactam-2H2O

Lactam-H2O

Hydrolactam-H2O

Hydrolactam

Bispyrrole

Pure pyrrole was added 1 mol% TEMED and 1 mol% APS 37 degree for 2 h

Figure 4.11S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with APS and TEMED at 37 ℃ under air.

212

Bispyrrole

Pyrrole-H2O Pyrrole-H2O Pure pyrrole was added 1 mMTMAO in H2O and reacted 37 degree 3hRatio as Pyrrole:TMAO=1:1

Lactam-H2O

Hydroxylactam-2H2O

Hydroxylactam-H2O

Lactam

Hydroxylactam

Figure 4.12S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with 1 mM TMAO at 37 ℃ 3 h.

Bispyrrole

Pyrrole-H2O

Pyrrole-H2O

Pure pyrrole was added 1 mMTMAO in H2O, 1mM EDTAin H2O and reacted 37 degree 3hRatio as Pyrrole:TMAO:EDTA=1:0.1:0.1

Hydroxylactam-H2O

Lactam

Hydroxylactam

Lactam-H2O

Hydroxylactam-2H2O

Figure 4.13S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with 1 mM TMAO/EDTA at 37 ℃ 3 h.

213

Pure pyrrole was added 1mM TEMPO in 10%ACN and reacted 37 degree 2 hRatio as Pyrrole:TEMPO=1:1

Bispyrrole

Pyrrole-H2O

Lactam-H2O

Hydroxylactam-2H2O

Lactam

Hydroxylactam-H2O Hydroxylactam

Matrix

Pyrrole-H2O

Figure 4.14S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with 1 mM TEMPO at 37 ℃ 2 h.

Bispyrrole

Pyrrole-H2O

Pyrrole-H2O

Lactam-H2OHydroxylactam-2H2O

Hydroxylactam-H2O

Lactam

Hydroxylactam

Pure pyrrole was added 1 mMTEMPO in 10%ACN, 1 mM EDTAin H2O and reacted 37 degree 2hRatio as Pyrrole:TEMPO:EDTA=1:1:1

Figure 4.15S MALDI-TOF spectrums of purified iso[4]LGE2-pyrrole reaction with 1 mM TEMPO/EDTA at 37 ℃ 2 h.

214

10ul 100mM 3-Ethyl-2,4-dimethylpyrrole in ACN +10ul ACN 25 degree for 3h

Dimer

Trimer

HN

H3C

HN

CH3

CH3

CH3CH3

CH3

HN

H3C

HN

CH3

CH3CH3

CH3

HN

CH3

CH3

CH3

10ul 100mM 3-Ethyl-2,4-dimethylpyrrole in ACN +10ul 100mM TEMPO in ACN 25 degree for 3h

DimerTrimer

HN

H3C

HN

CH3

CH3

CH3CH3

CH3 HN

H3C

HN

CH3

CH3CH3

CH3

HN

CH3

CH3

CH3

215

10ul 100mM 3-Ethyl-2,4-dimethylpyrrole in ACN +10ul 100mM TMAO in ACN 25 degree for 3h

Dimer

HN

H3C

HN

CH3

CH3

CH3CH3

CH3

Trimer

HN

H3C

HN

CH3

CH3CH3

CH3

HN

CH3

CH3

CH3

Figure 4.16S MALDI-TOF spectrums of 3-ethyl-2,4-dimethylpyrrole initiate reactions.

216

Pure 3-Ethyl-2,4-dimethylPyrrole -- Staring material

Figure 4.17S: NMR spectrums of 3-ethyl-2,4-dimethylpyrrole and 3-ethyl-1,2,4-trimethylpyrrole.

217

3-ethyl-1,2,4-trimethyl-1H-pyrrolein ACN for 2 h at R T

N

H3C CH3

CH3

Dimer

Trimer

3-ethyl-1,2,4-trimethyl-1H-pyrrolein ACN for 24 h at R T

N

H3C CH3

CH3

Dimer

Trimer

218

3-ethyl-1,2,4-trimethyl-1H-pyrrolein ACN was added TEMPO for 2 h at R T

N

H3C CH3

CH3Dimer

Trimer

3-ethyl-1,2,4-trimethyl-1H-pyrrolein ACN was added TEMPO for 24 h at R T

N

H3C CH3

CH3Dimer

Trimer

Figure 4.18S MALDI-TOF spectrums of 3-ethyl-1,2,4-trimethylpyrrole initiate reactions.

219

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 1000.0

0.5

1.0

1.5

2.0

2.5

3.0

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic

Equationy = A2 + (A1-A2)/(1 + (x/x0)^p)

Reduced Chi-Sqr

0.00429 0.00493

Adj. R-Square 0.9965 0.5946Value Standard Error

Iso4-HSA

A1 2.73774 0.03437A2 0.3593 0.03772x0 0.04611 0.00465p 1.06258 0.09919EC20 0.01251 --EC50 0.04611 0.00465EC80 0.16997 --

LGE2-BSA

A1 2.00993 0.04965A2 2.18793 0.02654x0 4.38542E-4 9.1443E11p 42.5833 1.36294E17EC20 4.24495E-4 --EC50 4.38542E-4 9.1443E11EC80 4.53053E-4 --

Iso[4]LGE2 Immunized Mouse Serum (Num 968)

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3 Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr

0.00562 0.03004


Iso4-HSA

A1 2.61284 0.04279A2 0.1448 0.04066x0 0.02219 0.00252p 1.02856 0.10652EC20 0.00576 --EC50 0.02219 0.00252EC80 0.0854 --

LGE2-BSA

A1 1.68854 0.07977A2 2.34502 0.09011x0 0.06851 0.05174p 1.80698 1.60749EC20 0.03181 --EC50 0.06851 0.05174EC80 0.14754 --


1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3 Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)


Model Logistic


Reduced Chi-Sqr

8.41644E-4 6.39738E-4


Iso4-HSA

A1 2.49696 0.01491A2 0.15149 0.01592x0 0.03961 0.00164p 1.19425 0.05244EC20 0.01241 --EC50 0.03961 0.00164EC80 0.12646 --

LGE2-BSA

A1 2.77897 0.00954A2 2.54044 0.05882x0 13.19208 8.6989p 1.14809 0.62089EC20 3.94379 2.16219EC50 13.19208 8.6989EC80 44.12786 52.61921

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 1000

1

2

3

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)


Model Logistic


Reduced Chi-Sqr0.01547 0.00292


Iso4-HSA

A1 1.8093 0.07646A2 0.24286 0.06278x0 0.00981 0.0029p 1.04583 0.28089EC20 0.00261 --EC50 0.00981 0.0029EC80 0.03693 --

LGE2-BSA

A1 2.68675 0.01923A2 2.4815 0.05261x0 3.79482 187001.28669p 11.33427 9.23029E6EC20 3.35794 168996.85846EC50 3.79482 187001.28669EC80 4.28854 638493.91753

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3 Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)


Model Logistic


Reduced Chi-Sqr

0.00157 0.00146


Iso4-HSA

A1 2.50431 0.02101A2 0.1267 0.02728x0 0.09768 0.00701p 0.87433 0.04871EC20 0.02001 --EC50 0.09768 0.00701EC80 0.47686 --

LGE2-BSA

A1 2.60962 0.01352A2 2.4795 0.03815x0 4.0171 325238.25016p 11.56137 7.97073E6EC20 3.56318 6072.41457EC50 4.0171 325238.25016EC80 4.52885 741060.46055

Figure 5.1S Cross-reactivity of iso[4]LGE2-HSA (■) with respect to LGE2-BSA (○) for immunized mice serum (104 dilution).

220

Figure 5.2S 20 plates (total 1920 wells) were done for the fusion step and 725 wells contained hybridoma cells.

224

2E8

(VL) 9G8 (VL)

12G4 (L)

15A5 (L)

18G7 (XXL)

4G5 (S)

5B7 (M)

8B3 (S)

10C11 (M)

16E4 (M)

19A4 (M)

Mice Serum

Iso4-BSA 0.336 2.826 2.557 0.271 2.528 0.123 0.12 0.112 0.136 0.12 0.119 2.825 Iso4-HSA 0.422 2.627 2.868 0.33 2.801 0.111 0.112 0.107 0.155 0.118 0.11 2.875 BSA 0.111 0.087 0.084 0.093 0.088 0.087 0.086 0.085 0.088 0.085 0.086 0.09 LGE2-BSA 0.238 0.105 0.106 0.199 0.835 0.087 0.085 0.084 0.103 0.087 0.088 0.193 LGE2-KLH 0.596 0.3 0.28 0.452 2.704 0.096 0.092 0.087 0.137 0.088 0.089 2.273 CEP-BSA 0.483 0.115 0.144 0.377 2.934 0.112 0.111 0.115 0.148 0.108 0.11 0.233

Clone 9G8(VL) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.101 0.139 0.341 1.721 2.901 2.866 LGE2-BSA 2.779 2.768 2.787 2.764 2.741 2.773 Clone 12G4(L) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.162 0.282 1.077 1.581 1.908 2.161 LGE2-BSA 1.671 1.642 1.642 1.618 1.984 1.99 18G7(XXL) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.158 0.28 0.796 1.039 1.098 1.23 LGE2-BSA 0.713 0.666 0.7 0.906 1.062 1.192 2E8 (VL) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.185 0.145 0.139 0.157 0.171 0.178 LGE2-BSA 0.141 0.132 0.135 0.154 0.165 0.183 15A4(L) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.194 0.189 0.192 0.208 0.209 0.211 LGE2-BSA 0.195 0.210 0.208 0.214 0.205 0.216 4G5(S) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.091 0.087 0.088 0.089 0.087 0.092

0

0.5

1

1.5

2

2.5

3

3.5

Iso4-BSA

Iso4-HSA

BSA

LGE2-BSA

LGE2-KLH

CEP-BSA

225

LGE2-BSA 0.086 0.085 0.086 0.085 0.085 0.089 5B7(M) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.091 0.091 0.087 0.091 0.091 0.091 LGE2-BSA 0.105 0.099 0.096 0.102 0.104 0.098 8B3(S) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.090 0.088 0.091 0.090 0.091 0.094 LGE2-BSA 0.094 0.091 0.094 0.091 0.090 0.101 10C11(M) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.114 0.113 0.109 0.112 0.114 0.117 LGE2-BSA 0.101 0.098 0.099 0.103 0.107 0.113 16E4(M) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.091 0.090 0.089 0.090 0.091 0.094 LGE2-BSA 0.093 0.095 0.094 0.094 0.094 0.095 19A4(M) 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 0.0286 μM 0.00572 μM Iso4-HSA 0.087 0.104 0.085 0.088 0.093 0.090 LGE2-BSA 0.089 0.092 0.091 0.089 0.090 0.094

1E-3 0.01 0.1 1 10 100

0

1

2

3

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

First Screening Clone 9G8 (VL)

Model Logistic


Reduced Chi-Sqr

0.00542 3.47E-4

Adj. R-Square 0.99695 -0.38874Value Standard Error

Iso4-HSA

A1 2.92998 0.06295A2 0.12987 0.05353x0 0.16781 0.01153p 1.87261 0.31819EC20 0.08004 --EC50 0.16781 0.01153EC80 0.35182 --

LGE2-BSA

A1 2.757 0.01347A2 2.778 0.01307x0 0.15186 24535.50489p 11.3928 3.02543E7EC20 0.13446 --EC50 0.15186 24535.50489EC80 0.17151 --

1E-3 0.01 0.1 1 10 1000

1

2

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

First Screening Clone 12G4 (L)

Model Logistic


Reduced Chi-Sqr

0.01697 7.05375E-4


Iso4-HSA

A1 2.16432 0.17644A2 -0.03979 0.26488x0 0.58803 0.2551p 0.77428 0.27782EC20 0.09814 --EC50 0.58803 0.2551EC80 3.52351 --

LGE2-BSA

A1 1.99 0.02656A2 1.64325 0.01531x0 0.03668 117253.60997p 16.22487 2.08308E8EC20 0.03368 --EC50 0.03668 117253.60997EC80 0.03996 --

1E-3 0.01 0.1 1 10 1000.0

0.5

1.0

1.5First Screening Clone 18G7 (XXL)

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr0.00555 0.002


Iso4-HSA

A1 1.16183 0.05626A2 0.10589 0.11033x0 1.08726 0.35461p 1.19937 0.42082EC20 0.34226 0.13992EC50 1.08726 0.35461EC80 3.45395 2.11388

LGE2-BSA

A1 1.20755 0.07748A2 0.67638 0.03667x0 0.0899 0.04122p 1.05988 0.46226EC20 0.02431 --EC50 0.0899 0.04122EC80 0.3325 --

Figure 5.3S Indirect and competitive ELISA tests of first screening clones. 9G8, 12G4, 18G7 showed best responses. 9G8 and 12G4 were picked for first round of subclone.

226

12G4 A2 A10 A11 A12 B2 B4 B5 B6 B8 B10 B12 C2 Iso4-BSA

2.620 2.639 2.660 2.600 1.843 2.562 2.523 2.577 2.601 2.568 2.572 2.674

Iso4-HSA

2.879 2.788 2.841 2.785 2.305 2.809 2.863 2.857 2.884 2.775 2.825 2.833

LGE2-BSA

0.111 0.134 0.147 0.145 0.100 0.151 0.121 0.157 0.147 0.172 0.166 0.163

Control 0.038 0.037 0.035 0.034 0.037 0.037 0.036 0.035 0.035 0.034 0.037 0.039 12G4 C3 C4 C7 C11 D6 D7 D8 D10 D11 E3 E10 F3 Iso4-BSA

2.861 2.760 2.708 2.905 2.808 2.920 2.800 2.909 2.978 2.880 2.914 2.998

Iso4-HSA

2.817 2.842 2.876 2.809 2.848 2.873 2.813 2.828 2.828 2.860 2.863 2.966

LGE2-BSA

0.119 0.136 0.106 0.228 0.147 0.120 0.164 0.152 0.149 0.101 0.157 0.127

12G4 F4 F5 F10 F11 G1 G3 G4 G6 H2 H10 9G8 12G4 Iso4-BSA

2.773 2.676 2.793 2.812 2.805 2.857 2.790 2.904 2.688 2.795 2.877 2.802

Iso4-HSA

2.797 2.761 2.815 2.777 2.762 2.820 2.863 2.847 2.804 2.940 2.793 2.964

LGE2-BSA

0.117 0.134 0.150 0.132 0.157 0.117 0.149 0.152 0.129 0.163 0.126 0.169

Control 0.037 0.035 0.036 0.037 0.037 0.036 0.035 0.035 0.037 0.035 0.037 0.036

Figure 5.4S Indirect ELISA tests of first subclones of 12G4, and all 34 subclones showed strong specificity response.

0

0.5

1

1.5

2

2.5

3

3.5

First Subclones of 12G4

Iso4-BSA

Iso4-HSA

LGE2-BSA

227

12G4 Subclone supernatant

A2 B2 B5 C3 C7 E3 F3 F4 G3 H2 12G4 control

Iso4-BSA 2.909 2.124 2.866 2.850 2.837 2.904 2.946 2.861 2.946 2.946 2.877 0.095 Iso4-HSA 2.992 2.721 2.992 2.998 2.975 2.991 2.998 2.999 2.968 2.992 2.984 0.116 LGE2-BSA 0.139 0.098 0.136 0.121 0.119 0.114 0.156 0.129 0.117 0.135 0.162 0.089 12G4 Subclone supernatant 1 to 10 Dilution

A2 B2 B5 C3 C7 E3 F3 F4 G3 H2 12G4 control

Iso4-BSA 2.452 0.836 2.445 2.262 2.026 2.120 2.627 2.525 2.368 2.582 2.625 0.108 Iso4-HSA 2.801 1.336 2.881 2.711 2.799 2.753 2.916 2.884 2.852 2.966 2.869 0.094 LGE2-BSA 0.099 0.096 0.096 0.101 0.101 0.098 0.105 0.098 0.097 0.105 0.112 0.093

Clone F3 89.33 μM

17.86 μM

3.572 μM

0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

9.15x10-6 μM

Iso4-HSA 0.273 1.236 2.609 2.894 2.898 2.912 2.923 2.934 2.942 2.956 2.964 LGE2-BSA 2.583 2.691 2.828 2.924 2.964 2.968 2.969 2.972 2.961 2.962 2.920

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 1000

1

2

3

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

First SubClone F3 From 12G4 (IgG1)

Model Logistic


Reduced Chi-Sqr

4.57502E-4 3.11822E-4


Iso4-HSA

A1 2.93245 0.00778A2 0.12189 0.03423x0 13.5591 0.38499p 1.52238 0.05758EC20 5.45454 0.196EC50 13.5591 0.38499EC80 33.70573 1.7493

LGE2-BSA

A1 2.96075 0.00707A2 2.53953 0.04421x0 8.98885 3.02324p 0.9191 0.19181EC20 1.98906 0.49487EC50 8.98885 3.02324EC80 40.62194 24.45795

00.5

11.5

22.5

33.5

A2

B2

B5

C3

C7 E3 F3 F4 G3

H2

12G

4A

2 10

times

dilu

tion

B2

10tim

es d

ilutio

nB

5 10

times

dilu

tion

C3

10tim

es d

ilutio

nC

7 10

times

dilu

tion

E3 1

0tim

es d

ilutio

nF3

10t

imes

dilu

tion

F4 1

0tim

es d

ilutio

nG

3 10

times

dilu

tion

H2

10tim

es d

ilutio

n12

G4

10tim

es d

ilutio

n

First subcloning of 12G4 (10 subclones)

Iso4-BSA

Iso4-HSA

LGE2-BSA

228

Clone F4 89.33 μM 17.86 μM 3.572 μM 0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

9.15x10-6 μM

Iso4-HSA 0.103 0.123 0.372 1.81 2.411 2.672 2.867 2.701 2.483 2.567 2.828 LGE2-BSA 1.938 2.073 1.943 2.068 2.247 2.53 2.594 2.602 2.538 2.462 2.484

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3

First SubClone F4 From 12G4 (IgG1)

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr

0.01975 0.00443


Iso4-HSA

A1 2.66941 0.05594A2 0.07805 0.10931x0 1.06241 0.17855p 1.53969 0.34607EC20 0.43178 0.0931EC50 1.06241 0.17855EC80 2.61408 0.79247

LGE2-BSA

A1 2.53988 0.0297A2 1.9952 0.03776x0 0.13939 0.042p 1.71788 1.08487EC20 0.0622 --EC50 0.13939 0.042EC80 0.31239 --

Clone H2 89.33 μM 17.86 μM 3.572 μM 0.7144

μM 0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

9.15x10-6 μM

Iso4-HSA 0.152 0.528 1.923 2.821 2.823 2.935 2.978 2.894 2.799 2.908 2.97 LGE2-BSA 2.403 2.441 2.514 2.687 2.806 2.86 2.98 2.945 2.974 2.998 2.976

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3

First SubClone H2 From 12G4 (IgG1)

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr

0.00413 5.99668E-4


Iso4-HSA

A1 2.9075 0.0239A2 0.1184 0.07811x0 5.41547 0.46265p 1.50213 0.16144EC20 2.15194 0.199EC50 5.41547 0.46265EC80 13.62829 2.18571

LGE2-BSA

A1 2.98294 0.01396A2 2.36034 0.03844x0 0.66943 0.22011p 0.57159 0.09008EC20 0.05921 --EC50 0.66943 0.22011EC80 7.56842 --

Clone E3 17.86

μM 3.572 μM

0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

9.15x10-6 μM

Iso4-HSA 0.112 0.405 1.466 1.721 1.614 1.572 1.651 1.617 1.733 LGE2-BSA 1.852 1.672 1.657 1.699 1.888 1.907 1.953 1.867 1.9

229

1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2First SubClone E3 From 12G4 (IgG1)

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr

0.00427 0.00558


Iso4-HSA

A1 1.65156 0.02702A2 0.10152 0.07127x0 1.84199 0.25133p 2.14595 0.34992EC20 0.96545 0.16588EC50 1.84199 0.25133EC80 3.51435 0.60774

LGE2-BSA

A1 1.90675 0.03735A2 1.72 0.04313x0 0.03357 45925.02555p 13.68614 1.16874E8EC20 0.03034 --EC50 0.03357 45925.02555EC80 0.03715 --

Clone 12G4 89.33 μM 17.86 μM 3.572 μM 0.7144

μM 0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

9.15x10-6 μM

Iso4-HSA 0.326 1.209 2.303 2.645 2.934 2.928 2.931 2.899 2.839 2.875 2.882 LGE2-BSA 2.557 2.599 2.571 2.864 2.878 2.896 2.921 2.936 2.949 2.816 2.859

1E-6 1E-5 1E-4 1E-3 0.01 0.1 1 10 1000

1

2

3

Iso4-HSA LGE2-BSA

12G4 (IgG1)

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr

0.00316 0.002


Iso4-HSA

A1 2.89609 0.02195A2 -0.11517 0.18039x0 14.15572 2.45868p 0.96226 0.10066EC20 3.35167 0.37545EC50 14.15572 2.45868EC80 59.78639 18.25262

LGE2-BSA

A1 2.89357 0.0169A2 2.57567 0.03162x0 0.886 4884.16907p 10.57528 270803.30233EC20 0.77715 --EC50 0.886 4884.16907EC80 1.0101 --

Clone A2 17.86

μM 3.572 μM

0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

Iso4-HSA 0.408 1.937 2.398 2.436 2.417 2.380 2.598 2.436 2.379 LGE2-BSA 2.383 2.304 2.324 2.368 2.365 2.288 2.372 2.415 2.362 Clone B2 17.86 μM 3.572 μM 0.7144

μM 0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

Iso4-HSA 0.188 1.164 2.145 2.187 2.230 2.183 2.368 2.086 LGE2-BSA 2.117 2.043 2.071 2.076 2.105 2.101 2.309 1.648 Clone B5 17.86

μM 3.572 μM

0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

Iso4-HSA 0.178 0.972 1.992 2.039 2.116 2.018 2.208 2.223 2.023 LGE2-BSA 2.131 1.998 1.995 1.969 2.077 2.055 2.123 2.231 1.983 Clone C3 17.86

μM 3.572 μM

0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

Iso4-HSA 0.897 2.105 2.244 2.246 2.270 2.224 2.248 2.391 2.232 LGE2-BSA 2.422 2.265 2.286 2.334 2.291 2.189 2.199 2.312 2.199

230

Clone C7 17.86 μM

3.572 μM

0.7144 μM

0.1429 μM

2.86x10-2

μM 5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

Iso4-HSA 0.647 2.103 2.445 2.473 2.604 2.556 2.657 2.641 2.458 LGE2-BSA 2.451 2.464 2.469 2.484 2.555 2.573 2.632 2.475 2.559 Clone G3

17.86 μM

3.572 μM

0.7144 μM

0.1429 μM

2.86x10-

2μM 5.72x10-

3 μM 1.14x10-

3 μM 2.28x10-

4 μM 4.58x10-

5 μM 9.15x10-

6 μM 1.83x10-

6 μM 3.66x10-

7 μM Iso4-HSA

1.242 2.461 2.796 2.793 2.878 2.910 2.868 2.820 2.956 2.882 2.740 2.685

LGE2-BSA

2.520 2.408 2.455 2.641 2.567 2.611 2.644 2.743 2.642 2.602 2.460 2.566

Figure 5.5S Indirect and competitive ELISA tests of 10 clones from 12G4, and 12G4 F3 was chose for next subclone.

12G4 F3 Subclone supernatant A4 A6 B3 B6 C1 C4 C6 D1 control Iso4-BSA 2.962 2.852 2.890 2.672 2.877 2.733 2.906 2.834 0.096 Iso4-HSA 2.869 2.816 2.867 2.799 2.859 2.729 2.867 2.851 0.093 LGE2-BSA 0.203 0.116 0.173 0.106 0.144 0.106 0.201 0.178 0.087 12G4 F3 Subclone supernatant 1 to 10 Dilution

A4 A6 B3 B6 C1 C4 C6 D1 control

Iso4-BSA 2.907 2.467 2.781 1.996 2.803 2.128 2.902 2.790 0.100 Iso4-HSA 2.958 2.670 2.788 2.428 2.781 2.509 2.817 2.849 0.092 LGE2-BSA 0.123 0.100 0.107 0.093 0.111 0.095 0.117 0.114 0.091 12G4 F3 Subclone supernatant D4 E1 E4 F4 F5 G2 G4 G7 control Iso4-BSA 2.748 2.735 2.729 2.749 2.779 2.673 2.571 2.702 0.092 Iso4-HSA 2.907 2.875 2.848 2.838 2.814 2.827 2.774 2.830 0.089 LGE2-BSA 0.187 0.190 0.185 0.211 0.198 0.149 0.123 0.199 0.095 12G4 F3 Subclone supernatant 1 to 10 Dilution

D4 E1 E4 F4 F5 G2 G4 G7 control

Iso4-BSA 2.700 2.668 2.650 2.713 2.751 2.657 2.373 2.676 0.093 Iso4-HSA 2.986 2.937 2.828 2.905 2.860 2.801 2.751 2.881 0.092 LGE2-BSA 0.119 0.128 0.129 0.124 0.130 0.112 0.106 0.125 0.099

0

0.5

1

1.5

2

2.5

3

3.5

A4 A6 B3 B6 C1 C4 C6 D1 D4 E1 E4 F4 F5 G2 G4 G7

16 Secondary SubClones From 12G4 F3 (IgG)

Iso4-BSA

Iso4-HSA

LGE2-BSA

231

Figure 5.6S Indirect ELISA tests of 16 secondary subclones from 12G4 F3.

12G4 F3 SubClone

Iso4-BSA Iso4-HSA Iso4-KLH BSA KLH HSA LGE2-BSA CEP-BSA CEP-HSA

C1 2.732 2.971 2.643 0.105 0.110 0.106 0.149 0.146 0.147 D1 2.596 2.809 2.580 0.097 0.101 0.097 0.149 0.135 0.155 G4 2.533 2.815 2.386 0.093 0.098 0.093 0.110 0.113 0.112

12G4 F3 SubClone 10time Dilution

Iso4-BSA Iso4-HSA Iso4-KLH BSA KLH HSA LGE2-BSA CEP-BSA

CEP-HSA

00.5

11.5

22.5

33.5

16 Secondary SubClones From 12G4 F3 (IgG) (10 Times Dilution)

Iso4-BSA

Iso4-HSA

LGE2-BSA

0

0.5

1

1.5

2

2.5

3

3.5C1 D1 G4 Secondary Subclones from 12G4 F3

C1

C1 10timeDilutionD1

D1 10timeDilutionG4

G4 10timeDilution

232

C1 10time Dilution 2.157 2.635 1.777 0.102 0.104 0.105 0.107 0.103 0.102 D1 10time Dilution 2.370 2.839 2.057 0.096 0.098 0.094 0.106 0.102 0.105 G4 10time Dilution 1.576 2.422 1.338 0.100 0.106 0.100 0.103 0.099 0.103

Clone C1 446.65 89.33 μM 17.86 μM 3.572 μM 0.7144 μM 0.1429 μM 2.86x10-2 μM 5.72x10-3 μM 1.14x10-3 μM

Iso4-HSA 0.167 0.866 2.494 2.67 2.603 2.603 2.511 2.476 2.543 LGE2-BSA 2.725 2.727 2.725 2.662 2.679 2.629 2.573 2.545 2.569

0

0.5

1

1.5

2

2.5

3

3.5

C1 D1 G4 Secondary Subclones from 12G4 F3

C1

D1

G4

0

0.5

1

1.5

2

2.5

3

C1 D1 G4 Secondary Subclones from 12G4 F3

C1 10time Dilution

D1 10time Dilution

G4 10time Dilution

233

1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3

Iso4-HSA LGE2-BSA

Abs

orba

nce

(405

nm

)

Conc (mM)

Secondary SubClone C1 From 12G4 F3 (IgG1)

Model Logistic


Reduced Chi-Sqr

0.00508 4.64133E-4


Iso4-HSA

A1 2.56737 0.02922A2 0.15447 0.07536x0 64.78795 6.16553p 2.71801 0.71018EC20 38.90331 8.28027EC50 64.78795 6.16553EC80 107.89513 9.84797

LGE2-BSA

A1 2.54534 0.02859A2 2.72758 0.01936x0 0.27338 0.2359p 0.5939 0.30393EC20 0.02649 --EC50 0.27338 0.2359EC80 2.82161 --

Clone D1 446.65 89.33 μM 17.86 μM 3.572 μM 0.7144

μM 0.1429 μM

2.86x10-2 μM

5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

Iso4-HSA 0.237 1.071 2.467 2.74 2.744 2.727 2.718 2.607 2.521 2.511 2.576 LGE2-BSA 2.768 2.757 2.674 2.709 2.762 2.721 2.661 2.618 2.581 2.548 2.583

1E-5 1E-4 1E-3 0.01 0.1 1 10 1000

1

2

3

Iso4-HSA LGE2-BSA

Secondary SubClone D1 From 12G4 F3 (IgG1)

Abs

orba

nce

(405

nm

)

Conc (mM)

Model Logistic


Reduced Chi-Sqr

0.01029 0.00111


Iso4-HSA

A1 2.64274 0.03628A2 0.18262 0.12464x0 66.95829 7.53202p 1.99096 0.46569EC20 33.37389 6.99503EC50 66.95829 7.53202EC80 134.33893 24.92572

LGE2-BSA

A1 2.56746 0.02426A2 2.73474 0.01579x0 0.01721 0.01361p 0.9968 0.68458EC20 0.00428 --EC50 0.01721 0.01361EC80 0.06916 --

Clone G4 446.65 89.33 μM 17.86 μM 3.572 μM 0.7144

μM 0.1429 μM

2.86x10-2 μM

5.72x10-3 μM

1.14x10-3 μM

2.28x10-4 μM

4.58x10-5 μM

Iso4-HSA 0.146 0.312 1.715 2.509 2.493 2.441 2.361 2.296 2.29 2.22 2.204 LGE2-BSA 2.617 2.603 2.513 2.536 2.464 2.434 2.36 2.354 2.273 2.232 2.248

1E-5 1E-4 1E-3 0.01 0.1 1 10 100

0

1

2

3

Abs

orba

nce

(405

nm

)

Conc (mM)

Iso4-HSA LGE2-BSA

Secondary SubClone G4 From 12G4 F3 (IgG1)

Model Logistic


Reduced Chi-Sqr

0.01528 6.5648E-4


Iso4-HSA

A1 2.35277 0.04418A2 0.17214 0.1278x0 26.115 5.41129p 2.40971 1.04015EC20 14.69069 2.18377EC50 26.115 5.41129EC80 46.42352 20.08087

LGE2-BSA

A1 2.15437 0.10782A2 2.68791 0.10761x0 0.14208 0.19755p 0.22635 0.11969EC20 3.10904E-4 --EC50 0.14208 0.19755EC80 64.92507 --

Figure 5.7S Indirect and competitive ELISA tests of final three candidates from 12G4 F3--C1 D1 G4.

Antigens Iso4-BSA

Iso4-HSA

Iso4-KLH

LGE2-BSA

BSA HSA KLH CEP-BSA

CEP-HSA

OHdiA-BSA

PBS

mAb 2.833 2.935 2.948 0.252 0.155 0.13 0.178 0.344 0.354 0.228 0.128

234

446.5 89.33

μM 17.86 μM

3.572 μM

0.7144 μM

0.1429 μM

2.86x10-

2 μM 5.72x10-

3 μM 1.14x10-

3 μM Iso4-HSA 0.138 0.243 1.13 2.117 2.4172 2.4184 2.2734 2.472055 2.407706 Iso4-KLH 0.253 1.464 2.543 2.631 2.532 2.5102 2.5508 2.396981 2.408779 LGE2-BSA 2.581 2.462 2.57154 2.599 2.309 2.449 2.4964 2.430872 2.461716 LGE2-KLH 2.3932 2.535 2.537 2.507 2.399 2.3921 2.4624 2.392691 2.363735 CEP-BSA 2.691 2.604 2.628 2.5248 2.307 2.4712 2.3494 2.339712 2.456225 CEP-HSA 2.45092 2.699 2.659 2.567 2.316 2.4944 2.3134 2.327914 2.366266 HSA 2.712 2.562 2.72636 2.678 2.515 2.4416 2.3346 2.351938 2.325125

Figure 5.8S Indirect and competitive ELISA tests of final anti-iso[4]LGE2 clone 12G4 F3 D1 in serum free media.

0

0.5

1

1.5

2

2.5

3

3.5 Purified Iso[4]LGE2 mAb from HL-1 SFM

Iso4 mAb 5 μg/ml

0

20

40

60

80

100

120

-3 -2 -1 0 1 2 3 4 5

Abs

orba

nce

(405

nm)

pmol/well

Anti-Iso[4]LGE2 mAb 12G4 F3 D1(IgG1) in HL-1 Serum Free Media

Iso4-HSA

Iso4-KLH

LGE2-BSA

LGE2-KLHCEP-BSA

CEP-HSA

HSA

235

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