DEVELOPMENT OF LANTHANIDE-TAGGED SUBSTRATES TOWARDS DETECTION OF PROTEASES BY INDUCTIVELY COUPLED PLASMA-MASS

SPECTROMETRY (ICP-MS)

by

Urja Lathia

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Chemistry

University of Toronto

© Copyright by Urja Lathia 2008

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Abstract Development of lanthanide-tagged substrates towards detection of proteases by

inductively coupled plasma-mass spectrometry (ICP-MS)

Masters of Science, 2008

Urja Lathia

Graduate Department of Chemistry, University of Toronto

Rapid, sensitive and quantitative assays for proteases are of great significance for drug

development and in diagnosis of diseases. Herein, we describe work towards a novel

assay for the multiplexed detection of proteases using ICP-MS. Protease substrates

were synthesized containing a diethylenetriaminepentaacetic acid (DTPA) ligand to

chelate lanthanide metal ions at the N-terminus, providing a distinct tag for each

substrate when complexed with a lanthanide metal. A biotin label was appended to the

C-terminus allowing separation of uncleaved peptide from the digestion. The enzymatic

activities can then be determined in a multiplexed fashion by detecting the lanthanide

signal of the peptide cleavage products by ICP-MS.

Biotinylated substrates synthesized include DTPA-Gln-Val-Tyr-Gly-Nle-Nle-Lys(biotin)-

amide for calpain-1, DTPA-Asp-Gln-Val-Asp-Gly-Lys(biotin)-amide for caspase-3 and

DTPA-Gly- Pro-Gln-Gly-Leu-Glu-Ala-Lys-Lys(biotin)-amide for MMP-9. The substrates

were loaded with terbium, holmium and praseodymium respectively. As a proof of

concept, α-chymotrypsin assays was carried out using DTPA-Asp-Leu-Leu-Val-Tyr-Asp-

Lys(Biotin) loaded with lutetium, as a substrate. Calpain-1 assays were also performed.

Parallel assays with commercially available fluorogenic substrates for both the enzymes

were performed for comparison.

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Acknowledgements I would like to take this opportunity to thank all the people here and in India who have helped make this thesis possible. Firstly, and above all I thank my supervisor, mentor and guide Prof. Mark Nitz for all his ideas and support that he always offered. I thank Dr. Vladimir Baranov for his assistance with my ICP- MS samples and for all the useful insight and wisdom he shared with me. I thank the entire Nitz group including Joanna Poloczek, Carmen Leung, Heather Griffiths, Anthony Chibba, Anthony Rullo, Grace Ng, Rodolfo Gomez, Guohua Zhang, Lehua Deng, Yedi Sun, Anna Gudmundsdottir, Michael Leipold, Caroline Paul and Richard Jagt. Special thanks to Joanna who helped me to learn to operate all the equipment including the HPLC, the fluorometer, the MALDI and was always there to help. Special thanks to Anthony Rullo who was kind enough to provide me with the coumarin fluorophore that he synthesized. Special thanks to our postdoctoral fellow Dr. Mike Leipold for useful discussions and all the ideas he had to offer. I thank Prof. Drew Woolley, Prof. Cynthia Goh, Prof. Deborah Zamble, Prof. Ron Kluger and Prof. Scott Tanner for sharing their equipment and lab space when I needed it. I thank Prof. Mitch Winnik and Dr. Olga Ornatsky for all their useful discussions and bright ideas. I would also like to thank the entire Chemistry department here at University of Toronto that has been so warm and welcoming. It’s given me some of the greatest friends who have made this place a new home for me. I thank Shreya and Janki, my sisters and only family I have in Toronto for their love and support. Special thanks to Parneet, a family friend. I thank my parents and family back home who have always been my greatest strength and support. I thank my friends Shreepal, Saurabh, Tanvi, Chaitri, Ashwini and Urvi in Bombay without whom I would not be the person I am.

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TABLE OF CONTENTS

1 Introduction

1.1 Proteases 1

1.2 Detection of proteases 1

1.3 Lanthanides 3

1.4 Inductively Coupled Plasma- Mass Spectrometry (ICP-MS) 3

1.5 Using ICP-MS with Lanthanides in Biological assays 4

1.6 Strategy for the on-bead protease assay 5

1.7 Strategy for the solution-phase protease assay 7

1.8 Proteases of interest 8

2 Materials 12

3 Methods

3.1 Synthesis of Lanthanide metal tagged substrates 13

3.1.1 Solid phase peptide synthesis 13

3.1.2 Coupling of 7-(Diethylamino)coumarin-3-carboxylic acid 13

3.1.3 Coupling of DTPA 13

3.1.4 Global Deprotection and purification of peptide substrates 14

3.1.5 Loading of substrates with lanthanide metal 15

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3.2 α-Chymotrypsin assays 16

3.2.1 On-bead assay Fluorescence assay for α-chymotrypsin 16

3.2.2 Solution-phase fluorescence assay 17

3.2.3 Solution-phase ICP-MS assay 17

3.3 Calpain-1 assays 18

3.3.1 FRET assay 18

3.3.2 ICP-MS assay 18

3.4 ICP-MS sample preparation 19

4 Results and Discussions

4.1 Synthesis of Lanthanide metal tagged substrates 20

4.2 α-Chymotrypsin assays 21

4.2.1 On-bead assay 21

4.2.2 Solution-phase fluorescence assay 24

4.2.3 Solution-phase ICP-MS assay 25

4.2.4 Fluorescence assay using Suc-AAPF-AMC 31

4.3 Calpain-1 assays 34

4.3.1 FRET assay 34

4.3.2 ICP-MS assay 36

4.3.3 Competition assay of Calpain-1 39

5 Conclusion and future work 42

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6 References 44

7 Appendices 48 7.1 HPLC traces 48

7.1.1 α-Chymotrypsin substrate with the fluorophore 48

7.1.2 α-Chymotrypsin substrate containing a DTPA tag and a biotin label 48

7.1.3 MMP-9 substrate containing a DTPA tag and a biotin label 49

7.1.4 Caspase-3 substrate containing a DTPA tag and a biotin label 49

7.1.5 Calpain-1 substrate containing a DTPA tag and a biotin label 49

vii

List of Figures

Introduction

Figure 1 Schematic diagram of the ICP- MS system 4

Figure 2 Design of the novel on-bead protease substrate containing a

DTPA tag at the N-terminus

and a photolabile linker at the C-terminus 6

Figure 3 Strategy for the on bead protease assay 7

Figure 4 Design of the novel protease substrate containing

a DTPA tag and a biotin label 7

Figure 5 Strategy for the solution phase protease assay 8

α-Chymotrypsin assays

Figure 6 Chemical structure of 7-(Diethylamino)coumarin-3-carboxylic acid 22

Figure 7 On-bead fluorescence α-chymotrypsin assay 23

Figure 8 Solution-phase fluorescence α-chymotrypsin assay 24

Figure 9 Solution-phase fluorescence α-chymotrypsin assay-

using different concentrations of α-chymotrypsin 24

Figure 10 Structure of α-chymotrypsin substrate 25

Figure 11 Optimization of the streptavidin beads suspension used for assays 26

Figure 12 Solution-phase ICP-MS α-chymotrypsin assay 26

Figure 13 α-Chymotrypsin assay with different concentrations of enzyme 27

Figure 14 α-Chymotrypsin assay under Single turnover conditions 28

viii

Figure 15 Streptavidin pull down experiment in 8M urea 29

Figure 16 α-Chymotrypsin assay in presence of EDTA 29

Figure 17 α-Chymotrypsin assay in presence of product 30

Figure 18 α-Chymotrypsin assay using Suc-AAPF-AMC as a substrate 32

Calpain-1 assays

Figure 19 FRET assay of Calpain-1 using H-Lys(FAM)-

Glu-Val-Tyr-Gly-Met-Met-Lys(DABCYL)-OH as a substrate 35

Figure 20 FRET assay using different concentrations of calpain-1 36

Figure 21 A comparison of a FRET assay of calpain1 to an ICP MS assay 37

Figure 22 ICP-MS assay using different concentrations of calpain-1 38

Figure 23 Calpain-1 assays under single turnover conditions 38

Figiure 24 Competition assay for calpain-1 40

ix

List of Tables

Table 1 Typical operating conditions of the ICP-MS instrument ELAN

DRCPlusTM (PerkinElmer SCIEX) 19

Table 2 Lanthanide-tagged substrates synthesized 20

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List of Abbreviations

ACN Acetanitrile

AMC 7-amino-4-methylcoumarin

BOC t-Butoxycarbonyl

DABCYL 4 - ((4 - (dimethylamino)phenyl)azo)benzoic acid

DCM Dichloro methane

DIEA Diisoproplyethylamine

DMF dimethyl formamide

DMSO Dimetyl sulfoxide

DTPA Diehtylenetriaminepentaacetic acid

EDTA ethylenediaminetetraacetic acid

EGTA ethylene glycol tetraacetic acid

ESI-MS Electrospray Mass spectrometry

FAM Carboxyfluorescein

Fmoc fluoren-9-ylmethoxycarbonyl

HBTU hexaflurophosphate

HCl Hydrochloric acid

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

HOBt 1-hydroxybenzotriazole 2-(1H-Benzotriazole-1-yl)-1,1,3,3-

tetramethyluronium

Ho Holmium

ICP-MS Inductively coupled Plasma mass-spectrometry

Ir Iridium

Ln Lanthanide

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Lu Lutetium

MALDI-MS Matrix-assisted laser desorption/ionization-Mass Spectrometry

NHS N- Hydroxysuccinamide

NMP N- methyl pyroliidone

ppb Parts per billion

Pr Praseodymium

SPPS solid phase peptide synthesis

Tb Terbium

TFA Tri fluoroacetic acid

TIS Tri isopropyl silane

TNBS 2,4,6- trinitrobenzene sulfonic acid

Tris tris(hydroxymethyl)aminomethane

UV ultra violet

1

1 Introduction

1.1 Proteases

Proteases are enzymes that cleave amide bonds in proteins and peptides. They are

found ubiquitously in all form of life1. Proteases determine the lifetime of proteins in an

organism and play essential roles in a multitude of biological processes1. For example,

there are the caspases, a class of cysteine proteases which originally were discovered

in cytokine processing, but now have well defined roles in programmed cell death. One

of many other examples is β-secretase which has the ability to cleave β-amyloid

precursor protein and is an important causative factor in Alzheimers disease. They are

known to be involved in a number of pathological conditions including cancer2, diseases

of the CNS3, cardiac diseases4 and numerous viral and infectious diseases5.

Proteases can be classified into 6 types based on their mechanism of action: serine

proteases, threonine proteases, cysteine proteases, aspartic acid proteases,

metalloproteases and glutamic acid proteases. In the studies undertaken we have used

enzymes from the serine protease and the cysteine protease classes.

1.2 Detection of proteases

Investigation of the pathological and physiological functions of proteases involves their

detection in complex tissue samples and other biological matrices.

In order to characterize the role of proteases in pathologies, the development of more

sensitive and reproducible methods to detect proteases is essential. Many assays have

been developed to study substrate specificity and to screen inhibitors of proteases

however they usually involve screening one protease at a time6. Activity assays for

proteases are commonly performed with substrates that have a fluorophore or a

2

chromophore at the C-terminus. The coumarin fluorophores including 7-amino-4-

carbomoylmethylcoumarin (ACC) and 7-amino-4-methylcoumarin (AMC) are commonly

used. After cleavage, the coumarin fluorophore is released into solution and its

maximum emission is shifted to longer7 wavelengths8, 9. Commonly used chromophores

in enzyme activity assays are nitrobenzene derivatives like p-nitrophenol and p-

nitroaniline10,11. After cleavage, the chromophore is released into solution its absorption

spectrum shifts allowing the products to be directly monitored.

Fluorescence resonance energy transfer (FRET) is also a commonly used method to

detect enzyme activities. FRET substrates contain a fluorophore and a quencher

attached to opposite ends of the peptide substrate. After enzymatic cleavage, the

quenching is eliminated and fluorescence is observed12, 13. Other methods for detecting

enzyme activity include calorimetric assays14, amperometric assays15, radioactive

assays16, 17, and chemiluminescent assays18.

Efforts are being made to design, high throughput and efficient multiplexed methods that

can be used to obtain extensive information from a single small sample19, 20, 21. This is

not only attractive due to economic reasons but also for recognizing characteristic signal

profiles associated with the relative changes in an entire set of analytes. Detecting

multiple proteases simultaneously would be beneficial in the diagnosis and the

understanding of a wide variety of diseases. This would include identifying the presence

of particular disease-related proteases as well as detecting overexpression of signature

proteases. Thus, development of multiplexed protease assays which are sensitive,

reproducible and have a large dynamic range would be advantageous for applications in

medicine and biology.

3

1.3 Lanthanide Metals

Lanthanides are located in the block 5d of the periodic table. They have very low

bioavailability due to their low solubility as fluoride and as phosphate salts, and the

elements in the group share similar chemical properties which make them ideal for use

as tags in biological assays. Lanthanides primarily occur in the oxidation state III and

can form stable complexes with ligands containing oxygen and nitrogen. Lanthanides

form thermodynamically and kinetically stable complexes with 1,4,7,10-

tetrakis(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DOTA)-based ligands22 and

diethylenetriaminepentaacetic acid (DTPA)-based ligands23. DTPA was used as the

ligand in this study.

There are 24 lanthanide elements with 54 stable isotopes. A very well resolved mass

spectrum of the lanthanides can be obtained with Inductively Coupled Plasma-Mass

Spectrometry (ICP-MS)24 which has a large linear dynamic range (9 orders of

magnitude) and an exceptional sensitivity (sub-part per trillion, or attomole/microlitre

detection)25

1.4 Inductively Coupled Plasma Mass Spectrometry (ICP-MS)

The basic principle of an ICP-MS system is the use of plasma at a high temperature

which can generate positive ions. The plasma is formed using a combination of argon

gas and a radiofrequency generator.

The instrument consists of a spray chamber and a nebulizer into which the sample is

introduced in a liquid form. An aerosol is formed that is introduced into the plasma by the

sample injector. In the plasma the sample is atomized and converted to a gas. An ICP-

MS system for trace metal detection usually uses quadrapole mass filters as analyzers

4

and electron multipliers as the detection device26. The ultratrace detection limit of the

ICP- MS comes from the number of positive ions that are generated and transported to

the detector. The figure below shows a schematic diagram of the instrumentation.

Figure1. Schematic diagram of the ICP-MS system

The advantages of using ICP-MS as a detection method for elemental tags includes low

detection limits, large dynamic range and very good spectral resolution24 which makes it

a promising multiplexing tool in analytical chemistry.

1.5 Using ICP-MS with Lanthanides in Biological assays

The goal of our work presented here was to design a multiplexed protease assay using

lanthanide tags which can be detected by ICP-MS. Protease substrates, which are

essentially small peptide sequences known to be specifically cleaved by the protease of

interest can be tagged with a lanthanide metal. This can be achieved by attaching a

DTPA ligand to the N-terminus of the peptide, and then loading it with the desired

lanthanide metal.

Keeping in mind the number of stable lanthanide isotopes (54), this assay design can be

used for the multiplexed detection of proteases by using orthogonal protease substrates

5

each holding a different lanthanide metal ion isotope. This would allow for the

simultaneous detection of multiple proteases in a single sample. There were two

approaches taken to do this; 1) an on-bead protease assay which would involve the Ln-

tagged substrate directly attached to a solid support, and 2) a solution-phase protease

assay which would involve the addition of a biotin label to the C-terminus of the Ln-

tagged substrates allowing separation of cleaved from uncleaved peptides with

streptavidin.

There have been previous reports where elemental tagging has been used to detect

proteins27, 28, 29, 30, but to the best of our knowledge this is the first example of using

lanthanide metals as tags to develop multiplexed protease assays.

1.6 Strategy for the on-bead protease assay

Commercially available TentaGel beads are an ideal solid support for synthesis of

peptides. TentaGel resins are grafted co-polymers consisting of a low cross-linked

polystyrene matrix on which polyethylene is grafted. They are ideal for solid supported

assays of proteases 31 because they are functionalized with amine groups which allows

for SPPS to be carried out directly on the beads.

For our application protease substrates containing a metal chelator at the N-terminus

were synthesized on the beads (0.26 mmole/g loading, 10 µm beads) and a photolabile

linker was introduced at the C-terminus of the peptide sequence to allow the peptide to

be liberated from the bead to check its quality (Figure 2).

6

Figure 2: Design of the novel on-bead protease substrate containing a DTPA tag at the N terminus and a photolabile linker at the C-terminus.

An assay can be constructed so that enzyme incubation of the beads with the protease

leads to cleavage of the substrate, releasing the lanthanide complex from the beads into

solution. Analysis of the solution by ICP-MS would show a difference in metal ions

present, indicating the efficiency of the cleavage.

A multiplex assay can be achieved using different elements for different substrates on

different beads, thus enabling detection of multiple enzymes in biological samples by

monitoring the presence of the released elements. This method can also be used to

conduct substrate specificity studies for new proteases being discovered.

The focus was to explore the cleavage of a lanthanide-tagged substrate. An on-bead

assay was designed (Figure 3) for α-chymotrypsin using Asp-Leu-Leu-Val-Tyr-Asp as

the substrate. It was synthesized on a TentaGel support with a DTPA ligand containing

lutetium at the N-terminus and a photolabile linker at the C-terminus.

7

Figure 3. Strategy for the on-bead protease assay.

1.7 Strategy for the solution-phase assay

Protease substrates that contain a biotin label32 at the C-terminus and a DTPA tag at the

N-terminus to which a lanthanide metal can be chelated were synthesized. This

effectively produced a metal tagged protease substrate (Figure 4).

Figure 4. Design of novel protease substrates containing a DTPA tag at the N terminus and a biotin label at the C-terminus

On enzymatic cleavage the DTPA-lanthanide complex would be separated from the C-

terminal biotin label. Streptavidin that is conjugated to agarose beads can be used to

pull down the remaining uncleaved substrate molecules since they contain a biotin

label32. The agarose beads can merely be spun down by the use of a centrifuge and the

8

metal content in the supernatant can be analyzed by ICP-MS (Figure 5). The metal

content would directly correspond to the identity and the quantity of the protease.

Figure 5. Strategy for the solution phase protease assay

A multiplex protease assay similar to the on-bead assay can be designed in the solution-

phase. Orthogonal metal tagged substrates for various enzymes can be used to detect

multiple proteases in a single sample by ICP-MS. The protease can be identified by the

substrate that was cleaved, which would directly correspond to the metal that shows

increase in signal. Given the sensitivity of ICP-MS, this method can also be quantitative

and could be applied in several ways including substrate specificity studies of proteases,

inhibitor screening and detection of proteases in biological lysates.

1.8 Proteases of interest

MMPs are a large class of proteases known to play important roles in the early stages of

progression and metastasis of cancers as well being over expressed in various

conditions like arthritis6. Caspases, are predominantly involved in apoptosis and

increased levels are seen in cellular degenerative diseases like ischemic damage and

9

neurodegenerative disorders. Activation of caspases can also serve as therapy to kill

cells in proliferative diseases like cancer33. Calpains are another class of proteases that

have potential roles in apoptosis and thus, in pathological events causing neurological

deficits34. The biological roles of these various enzymes make them interesting

candidates for our study, since detection of these enzymes may find application in the

diagnosis of disease and in the development of new therapeutics for various disorders.

Serine proteases: As the name suggests, these proteases contain a serine residue in

the active site. Chymotrypsin-like serine proteases, which are the most abundant in

nature, have His-Asp-Ser as their catalytic triad35. Other major clans include subtilisin-

like proteases that have Asp-His-Ser as the catalytic triad; the carboxypeptidase Y-like

proteases containing Ser-Asp-His as the catalytic triad and the Clp proteases which

have Ser-His-Asp as the catalytic triad35. In the study presented we used α-

chymotrypsin from bovine pancreas, which hydrolyses amide bonds C-terminal to

aromatic amino acid residues. The sequence for the α-chymotrypsin substrate

synthesized was based on these properties.

Cysteine proteases: Cysteine proteases such as papain, cathepsins, caspases and

calpains, have active sites that involve a nucleophilic cysteine thiol.

In our study we were interested in the calpains which are a family of calcium dependent

cysteine proteases. Calpains have been thought to have roles in fibroblast motility and

platelet activation36. Additionally, they cleave several cytoskeletal proteins indicating that

calpains play a role in the cytoskeletal architecture and interact with the plasma

membrane36. Due to these functions, they are known to have potential roles in

apoptosis37. They exist as two isoforms: calpain-1 and calpain-2, also called µ- and m-

calpain, that share a common 28K regulatory subunit. Differing calcium concentrations

are required to activate the two isoforms; i.e. micromolar concentrations are required for

10

calpain-1 and millimolar concentrations are required for calpain-2. Calpain-1 was used in

the study reported here. The substrate synthesized for this protease was based on one

of its natural specific substrates which is an α-spectrin protein found in the

hippocampus38.

Caspases are another broad class of cysteine proteases that play essential roles in

apoptosis, necrosis and inflammatory responses. There are at least 11 known caspases

(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 14). Members of the caspase family require an aspartate

residue in the P1 position and caspase-3 is known to cleave poly(ADP-ribose)

polymerase (PARP), which is its natural substrate39. Thus, a substrate for caspase-3

was synthesized based on these properties.

Matrix metalloproteinases (MMPs) are involved in the proteolysis of proteins found in

the extracellular matrix and are zinc dependant enzymes. MMPs are involved in many

pathological conditions including cancer40, as well as diseases of the CNS and immune

system41. MMPs are divided into categories including collagenases, stromelysins,

gelatinases and matrilysins on the basis of substrate specificity as well as on structure40.

The sequence of the synthesized substrate for MMP-9 (gelatinase B) was based on a

sequence previously reported in literature which contains the cleavable Gly-Leu bond12.

The proteases chosen for a multiplexed assay were calpain-1, caspase-3 and MMP-9.

These were chosen because they are available commercially and specific substrates for

these enzymes are known from literature. A distinct orthogonal substrate sequence was

chosen and synthesized for each enzyme such that there would be no cross reactivity.

For initial experiments, α-chymotrypsin was chosen. It is a serine protease, a class of

proteases that has been very widely studied and is very well characterized. In addition, it

11

is one of the cheaper and readily available protease which makes it a good candidate for

initial studies as a proof-of-concept.

12

2 Materials

Most of the protected amino acids as well as HOBt and HBTU were purchased from GL

Biochem (Shanghai). The protected amino acids used from GL Biochem were- Fmoc-

Asp(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Nle-OH,

Fmoc-Lys-OH, Fmoc-Leu-OH and Fmoc-Pro-OH. Fmoc-Gly-OH, Fmoc-Gln(trt)-OH and

Fmoc-L-Ala-OH were purchased from Anaspec Inc. Fmoc-Asp(OtBu)-OH was

purchased from Novabiochem. The biotin labeled lysine [Fmoc-Lys (biotin)-OH] was

purchased from EMD Biosciences. These were used without further purification. DTPA

was purchased from Sigma. Streptavidin Agarose beads suspension was purchased

from EMD Biosciences. α-Chymotrypsin from bovine pancreas Type II was purchased

from Sigma and was used without any further purification. Calpain-1, Human

Erythrocytes was purchased from Calbiochem and was used without any further

purification. Chymotrypsin fluorogenic substrate [Suc-AAPF-AMC] and calpain-1

fluorogenic substrate [H-Lys(FAM)-Glu-Val-Tyr-Gly-Met-Met-Lys(DABCYL)-OH] were

purchase from EMD Biosciences. NovaPEG Rink amide resin (capacity 0.67mmole/g)

was purchased from Nova Biochem. TentaGel M NH2 (capacity 0.26mmol/g) was

purchased from RAPP Polymere. Fmoc- Photolabile linker was purchased from

Advanced ChemTech. ICP-MS grade Baseline Hydrochloric acid (35%) was purchased

from Seastar Chemicals. The Iridium standard 1000 mg/L (1000pm) and the Multi

Element Solution Standard 1 were purchased from SpexCertiprep. All other reagents,

including the lanthanide salts, buffer salts and solvents were bought from Sigma. All

buffers were made using Milli-Q water (Millipore).

13

3 Methods

3.1 Synthesis of Lanthanide metal-tagged substrates

3.1.1 Solid Phase Peptide Synthesis

Enzyme substrates were synthesized using standard Fmoc SPPS as described below.

Peptides were manually synthesized on NovaPEG Rink amide resin (0.67 mmole/g

loading, 150 mg) or Tentagel M NH2 beads (0.26 mmole/g loading, 10 µm beads, 100

mg, for the on bead assays). Each amino acid was coupled in the same way using HOBt

(50.26 mg) and HBTU (141.07 mg) to generate the active ester in the presence of DIEA

(96.16 mg) using DMF (6 ml) as the solvent (resin: amino acid: HOBt: HBTU: DIEA-

1:4:4:4:8, 93 µmoles: 372 µmoles: 372 µmoles: 744 µmoles). Removal of the Fmoc

group was done using 20% piperidine in DMF (6 ml, 20 minutes). The resin was

washed with DMF (2 minutes, 5 ml each wash, 3 times). The amino acid, HOBt, HBTU

and DIEA were dissolved in DMF (6 ml) and added to the resin. For coupling Fmoc

Lys(biotin) OH, NMP was used as the solvent . Each residue was coupled for 40

minutes. Deprotection and coupling was confirmed using the TNBS test (a reagent used

to identify primary amines).

3.1.2 Coupling of 7-(Diethylamino)coumarin-3-carboxylic acid

DIC and NHS were used to attach the fluorophore to the N-terminus of the peptide. 7-

(Diethylamino)coumarin-3-carboxylic acid: DIC: NHS (6:3:3) was dissolved in DMF and

allowed to stand for 20 minutes to form the activate ester. The solution was added to the

resin and was coupled overnight.

3.1.3 Coupling of DTPA

NHS and HBTU were used to attach DTPA to the N-terminus of the peptide.

Concentrations used were, resin: DTPA: DIEA: HBTU: NHS (1: 14: 140:28:28). DTPA

14

(512.14 mg) and DIEA (2.15 ml) were added to DMF (4 ml) and it was boiled to dissolve

the DTPA. HBTU and NHS were dissolved in DMF (4 ml) which was added to the DTPA

solution after it had cooled to room temperature. This solution was then added to the

resin and coupled overnight.

3.1.4 Global deprotection and purification of peptide substrates

A global deprotection of the protected peptide substrates using TFA/water/TIS (95: 2.5:

2.5, 10 ml) was done. The resin was filtered and the resulting TFA solution was

concentrated under nitrogen. The peptide was precipitated from the TFA solution in cold

diethyl ether.

Purification was carried out using reverse phase HPLC. Peptides were loaded onto a

C18 reverse phase column in water or in 5% DMSO and solvents used were ACN +

0.1%TFA and water + 0.1% TFA.

Purification of α-chymotrypsin substrate [DTPA-Asp-Leu-Leu-Val-Tyr-Asp-

Lys(Biotin)-amide] :

The peptide was loaded in 5% DMSO and 95% reaction buffer. The gradient used to

elute the peptide was 5 minutes 2% ACN, 45 minutes 50% ACN, 60 minutes 100%

ACN. The α- chymotrypsin substrate eluted at 31% ACN.

Purification of caspase-3 substrate [DTPA- Asp-Glu- Val- Asp-Gly- Lys(biotin)-

amide]:

The peptide was loaded in water. The gradient used to elute the peptide was 5 minutes

2% ACN, 65 minutes 50% ACN, 70 minutes 100% ACN. The caspase-3 substrate was

eluted at 22% ACN.

Purification of calpain-1 substrate [DTPA- Glu-Val-Tyr-Gly-Nle-Nle- Lys(biotin)-

amide]

15

The peptide was loaded in water. The gradient used to elute the peptide was 5 minutes

2% ACN, 60 minutes 50% ACN, 70 minutes 100% ACN. The calpain-1 substrate was

eluted at 32% ACN

Purification of MMP-9 substrate [DTPA Gly-Pro-Gln-Gly-Leu-Glu-Ala-Lys-

Lys(Biotin)-amide]

The peptide was loaded in water. The gradient used to elute the peptide was 5 minutes

2% ACN, 15 minutes 15% ACN, 60 minutes 35% ACN, 70 minutes 50% ACN, 75

minutes 100% ACN. The MMP-9 substrate was eluted at 21% ACN.

In the case of the TentaGel beads the peptide remained on the beads and the beads

were washed with DMF (2 minutes, 6 ml, 5 times).

3.1.5 Loading the substrate with a lanthanide metal

The purified peptide or the peptide supported on TentaGel beads was incubated with an

excess of lanthanide salt (approximately 3 mM) in 0.4 M ammonium acetate buffer pH 6

at room temperature. Desalting was done using a C18 column that was pre washed with

10 mM ammonium citrate buffer pH 6 (100 ml) and water (100 ml). The peptide was

loaded on the pre washed column. It was washed with ammonium citrate buffer pH 6

(100 ml) and water (100 ml) and then the peptide was eluted in 50% ACN. The solution

was frozen and lyophilized.

The substrate containing the fluorophore was quantified using UV spectrometry. While

the lanthanide-tagged substrates were quantified by ICP-MS using a Multi Element

solution standard (1 ppb)

16

3.2 α-Chymotrypsin assays

Chymotrypsin was reconstituted in 1 mM HCl containing 2 mM CaCl2. Aliquots were

kept at -20º C and were used within a week of reconstitution according to the

manufacturer’s instructions.

3.2.1 On-Bead Fluorescence assay for α-chymotrypsin

TentaGel beads with the α-chymotrypsin fluorogenic substrate (7-

(Diethylamino)coumarin-3-carboxylic acid -Asp-Leu-Leu-Val-Tyr-Asp) synthesized on

them were uniformly suspended in 0.1% tween 20. Bead suspension (2 mg/ 5 ml, 100 µl

~ 78 X 103 beads) was used in the assay. The suspension was centrifuged (13000 rpm,

45 minutes). Equal volumes of the supernatant were removed from the tubes (85 µl).

Assays were carried out in reaction buffer 100 mM tris pH 8, 10 mM CaCl2, 50 mM NaCl

at 25º C. An enzyme concentration (1 µM) was added to start the reaction and a blank

control containing the beads with no enzyme was prepared. Aliquots of 400 µl were

taken at desired times. Enzyme reaction was stopped by heating the aliquots at 100º C

for 15 minutes. Each aliquot was centrifuged (13000 rpm, 45 minutes) and the

fluorescence in the supernatant was read at an excitation wavelength of 420 nm and an

emission wavelength of 460 nm. A positive control containing the beads that were

subjected to UV light was prepared.

Positive control:

1% β mercaptoethanol, 2.5% HEPES buffer pH 7 in ACN: water (1:1) was added to the

beads. It was exposed to UV radiation (30 minutes) and centrifuged (13000 rpm, 45

minutes). The supernatant was removed (400 µl) for reading in the fluorometer.

Excitation wavelength was 420 nm and emission wavelength was 460 nm.

17

3.2.2 Solution-phase fluorescence assay

The α-chymotyrpsin substrate 7-(Diethylamino)coumarin-3-carboxylic acid -Asp-Leu-

Leu-Val-Tyr-Asp- Lys(biotin)-amide was synthesized and purified for a solution-phase

fluorescence assay. Assays were carried out in reaction buffer 100 mM Tris pH 8, 10

mM CaCl2, 50 mM NaCl. Substrate (10 µM) was incubated with α-chymotrypsin (400

nM, 40 nM and 4 nM) at 25º C. The total volume was 1350 µl. Reactions were started by

addition of the enzyme. Aliquots (450 µl) were taken at 0, 30 and 60 minutes; the

reaction was stopped by heating the aliquots at 100º C for 15 minutes. Streptavidin

agarose bead suspension (70 µl) was added, and shaken for 30 minutes at 25º C.

Samples were centrifuged (13000 rpm, 25 minutes). Fluorescence in the supernatant

(380 µl) was read. The fluorophore was excited at 420 nm and showed an emission

maximum at 460 nm. A blank control was run that was treated the same way but did not

contain any enzyme.

3.2.3 Solution-phase ICP-MS assay

The substrate synthesized was quantified using ICP-MS by the lutetium concentration

obtained. Substrate concentrations used were 2 µM and 10 µM and the assays were

carried out in reaction buffer -100 mM tris pH 8, 10 mM CaCl2, 50 mM NaCl at 25º C.

The reaction was stopped by heating the aliquots at 100º C for 20 minutes. The sample

was diluted 20 times (5 µl in 100 µl water). Streptavidin agarose bead suspension (70 µl)

was added to each sample and was shaken for 30 minutes at 25º C. Samples were

centrifuged (13000 rpm, 25 minutes) and the supernatant was diluted 5 times (35 µl in

175 ul water). Dilutions were changed according to substrate and enzyme

concentrations. A blank control was run that was treated the same way but did not

contain any enzyme. A sample containing the substrate and no enzyme, that was not

treated with streptavidin agarose served as a positive control.

18

3.3 Calpain-1 assays

3.3.1 FRET assay

The substrate was dissolved in DMSO (1 mg/ml) and aliquots were stored at

-20º C. Further dilutions of the substrate were made in the reaction buffer. The buffer

used was 50 mM tris HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 5 mM β-

mercaptoethanol and 5 mM CaCl238. The enzyme (200 nM or 500 nM) was added to the

substrate (10 µM) to start the reaction which was carried out at 25º C. A blank control

containing the substrate without any enzyme was run. 10 µl aliquots (at 1, 2, 3, 5, 10, 15,

20, 30, 60 minutes) were added to 90 µl of 8 M urea to stop the reaction and also to

provide a dilution to bring the signal to a detectable value. A black multi well plate was

loaded with sample (100 µl) and was read by a fluorescence plate reader. The excitation

wavelength was 490 nm and the emission wavelength was 518 nm. A sample that was

incubated overnight with calpain-1 served as a positive control.

3.3.2 ICP-MS assay

The substrate synthesized was quantified using ICP-MS by the terbium concentration

obtained. The buffer used was 50 mM tris HCl pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM

EGTA, 5 mM β-mercaptoethanol and 5 mM CaCl2. The enzyme (500 nM or 1 µM) was

added to the substrate (10 µM) to start the reaction and was incubated at 25º C. A blank

control containing the substrate without any enzyme was run. 5 µl aliquots (at 1, 2, 3, 5,

10, 15, 20, 30, 60 minutes) were added to 95 µl of 8 M urea (20 times diluted). A sample

containing the substrate and no enzyme, that was not treated with streptavidin agarose

served as a positive control. To the samples (100 µl) streptavidin agarose bead

suspension (70 µl) was added and reacted for 30 minutes at 25º C. Samples were

centrifuged (13000 rpm, 25 minutes) and the supernatant was diluted twice (76 µl

sample added to 76 µl water).

19

3.4 ICP-MS sample preparation

50 µl of diluted assay sample was added to 50 µl Ir standard (1 ppb in 10% HCl). Each

sample was run in triplicates. A Multi Element Solution Standard 1, diluted to 1 ppb was

used to quantify the samples. The parameters used for the ICP-MS are listed below

(Table 1).

Parameter Value Plasma power 1400 W Cross flow nebulizer, Ar flow 0.95 L min-1 Plasma gas, Ar flow 17 L min-1 Auxiliary gas, Ar flow 1.2 L min-1 CeO+/Ce+ ratio in 10% HCl <3% Typical sensitivity 1 ppb Ir in 10% HCl, typical 104 cps 1 ppb In in 10% HCl, typical 4 X104 cps

Table 1. Typical operating conditions of the ICP-MS instrument ELAN

DRCPlusTM (PerkinElmer SCIEX).

20

4 Results and discussions

4.1 Synthesis of Lanthanide metal-tagged substrates

Lanthanide-tagged substrates synthesized are shown below (Table 2).

Protease Lanthanide-tagged substrate α-Chymotrypsin Lu-DTPA-Asp-Leu-Leu-Val-Tyr~Asp-Lys(biotin)-amide

MMPs MMP-9 Pr-DTPA-Gly- Pro-Gln-Gly~Leu-Glu-Ala-Lys-Lys(biotin)-amide

Caspases Caspase-3 Ho-DTPA-Asp-Gln-Val-Asp~Gly-Lys(biotin)-amide

Calpains Calpain-1 Tb-DTPA-Gln-Val-Tyr~Gly-Nle-Nle-Lys(biotin)-amide

Table 2: Lanthanide-tagged substrates synthesized.

SPPS was used to synthesize the substrates and the purification was done on a C18

column using reverse phase HPLC. The peaks were collected and identified using

MALDI–MS.

Loading the peptides with the lanthanide metal

The complete loading of the peptide with metal, was monitored using Mass

Spectrometry. MALDI- MS was used but it was observed that partial dissociation of

DTPA-Ln coordination occurred during ionization and thus ESI-MS was used to confirm

complete Ln loading when the MALDI-MS spectrum was not quantitative.

When a commercially available C18 SepPak column was used for desalting the peptide,

high background lanthanide counts were obtained in the sample, and it was suggested

that the SepPak itself could be a source of Ln contamination. Prewashing the SepPak

and analysis of column washes showed significant residual Ln counts. A small C18

column attached to a peristaltic pump was set up. Ammonium citrate buffer pH 6 (10

mM) was used for pre-washing the column since citrate functions as a weak Ln metal

21

chelator that can help capture any unconjugated Ln ions, keeping the Ln ions in the

DTPA complex intact. The pump delivered the solvent at approximately 2 ml per minute.

After loading the column, it was washed extensively with ammonium citrate buffer (pH 6,

10 mM) and water. This setup efficiently removed the background problems due to

residual lanthanide ions.

4.2 α-Chymotrypsin assays

4.2.1 On-bead assay

The first approach to developing a multiplexed protease assay was to produce Ln-

tagged protease substrates on TentaGel beads. This would allow for orthogonal

protease substrates each holding a distinct tag, to be carried on different beads. This

would result in a very simple and a quick multiplexed protease assay.

As a proof-of-concept, a fluorogenic substrate for α-chymotrypsin using

(Diethylamino)coumarin-3-carboxylic acid (Figure 6) as a fluorophore attached to the N-

terminus of the peptide was developed. This enzyme was chosen because it is very well

characterized, relatively cheap and readily available.

Assays were initially done using the fluorescent labeled substrate, 7-

(Diethylamino)coumarin-3-carboxylic acid- Asp-Leu-Leu-Val-Tyr-Asp that was

synthesized on TentaGel beads (10 µm beads, 0.26 mmol/g loading) (Figure 7). For

quality control a photolabile linker was introduced before the sequence at the C-

terminus such that the peptide can be cleaved under UV light to release it from the bead

into solution. This allows the sequence to be confirmed using MALDI-MS and also

serves as a positive control in the assays. The product formed by enzymatic cleavage

would be released from the beads and the product was separated form the beads by

centrifugation. The supernatant was analyzed for the product by fluorescence.

22

Figure 6. Chemical structure of 7-(Diethylamino)coumarin-3-carboxylic acid

To allow for accurate pipeting a uniform suspension of the beads was required. The

suspension of beads was analyzed with optical microscopy and different surfactants and

sonication conditions were evaluated. Sodium dodecyl sulphate (10 mM, 100 mM) and

Igepal CO-520 (10 mM, 100 mM) with bath or probe sonication gave aggregated

samples. Tween 20 was tried at concentrations of 0.1%, 0.5%, 1%, 2%, 3%, 4% and 5%

v/v. At a low concentration of 0.1%v/v tween 20 with probe sonication (12watts) the

beads appeared to be uniformly suspended and no damage to the beads was observed.

These conditions were used for the experiments.

Since the background fluorescence signal from the assay was high, it was suggested

that the coumarin maybe nonspecifically adsorbed to the TentaGel beads and was being

released during the assay. Beads without any peptide were incubated with the

fluorophore under the same reaction conditions as were used to couple the coumarin to

the peptide to test for non specific binding or adsorption of the fluorophore and this was

not to be the case. Perhaps, the fluorophore molecules were trapped within the peptide

framework on the bead, producing a background signal. Thus before running assays,

the beads were washed extensively. But, washings with the reaction buffer at 40º C for 4

days did not seem to help. In view of the fact that the fluorophore dissolves in organic

solvents, washing several times with DCM and DMF was attempted. This appeared to

help to some extent.

23

The positive control was based on the peptide cleaved off the bead by UV light, which

may not allow complete release of the substrate into solution, thus the control may be an

underestimate of the total amount of peptide present.

Furthermore, compared to the positive controls run during the assays, only a small

fraction of the substrate was being cleaved. It would be expected that there is steric

hindrance due to the polymeric nature of the bead and thus the enzyme could not

access all of the sites of cleavage effectively. One solution to this problem would be to

block most of the hindered reaction sites on the TentaGel bead before carrying out

SPPS; this would lead to less peptide on the bead consequently less overcrowding and

perhaps a cleaner assay42. Increasing the distance of the substrate sequence from the

bead, via a spacer, may also be an effective way to prevent interference from the bead.

Figure 7. On-bead fluorescence α-chymotrypsin assay. Blank samples-pink (▪), Test samples

(with 1 µM chymotrypsin)-blue (♦). The reaction buffer used was100 mM tris pH 8, 10 mM CaCl2, 50 mM NaCl at 25º C. 400 µl of sample was withdrawn at each time point and the reaction was

stopped by heating at 100º C. Fluorescence in the supernatant was read at an excitation wavelength of 420 nm and an emission wavelength of 460 nm.

The observations showed that only less than 5% of the on-bead substrate was cleaved

after 18 hours of enzyme incubation at a relatively high enzyme concentration. Due to

this reason and the high backgrounds obtained the on-bead approach was not pursued

and a solution-phase assay was designed.

24

4.2.2 Solution-phase fluorescence assay

Similar to the on-bead assay, initial experiments were carried out using 7-

(Diethylamino)coumarin-3-carboxylic acid- Asp-Leu-Leu-Val-Tyr-Asp-Lys(Biotin) as a

fluorescence substrate for α-chymotrypsin. The substrate synthesized contained a biotin

label at one end, this would allow for the separation of uncleaved substrate molecules by

using streptavidin protein. This preliminary assay was to confirm that the sequence was

cleaved by the enzyme and that the biotin pull down was quantitative.

Figure 8. Solution-phase fluorescence α-chymotrypsin assay. Substrate concentration used was

10 µM. Blank samples (pink, ■), 40 nM α-chymotrypsin (blue,♦)

The fluorescence intensity was approximately 40 times higher at 150 minutes compared

to the blank sample when a substrate concentration of 10 µM and an enzyme

concentration of 40 nM were used (Figure 8).

Figure 9. Solution-phase fluorescence α-chymotrypsin assay - Using different concentrations of enzyme. Substrate concentration was 10 µM. α-Chymotrypsin concentrations used were 4 nM,

40 nM and 400 nM.

0

50000

100000

150000

200000

250000

300000

350000

400000

0 10 20 30 40 50 60

Time (mins)

fluor

osen

ce in

tens

ity

chymotrypsin 4nM

chymotrypsin 40nM

chymotrypsin 400 nM

No chymotrypsin

25

With increasing concentrations of α-chymotrypsin an increase in peptide cleavage was

observed (Figure 9). These results indicate that the sequence was cleaved by α-

chymotrypsin and that the biotin pull down format lead to reproducible results. The on-

bead α-chymotrypsin assay had a high background signal and less than 5% of the

substrate was converted to the product with a high enzyme concentration of 1 µM.

However, with the solution phase assay the background signal was low and a small

enzyme concentration of 4 nM could be detected, making the assay considerably more

sensitive. Given the positive outcome of the fluorescence assay a lutetium chelated

substrate was synthesized to conduct ICP-MS based assays.

4.2.3 Solution-phase ICP-MS assay

The sequence Asp-Leu-Leu-Val-Tyr-Asp-Lys(biotin)-amide containing a biotin label and

a DTPA tag was synthesized as a α-chymotrypsin substrate (Figure 10). After synthesis

and purification the DTPA was chelated to lutetium to give a lanthanide tagged

substrate.

HON

N

ON

O

OH

O

HO

O

NH

OHO

HN

O

NH

OHN

O

NH

OHN

O

NH

O

OOH

HOO

NH2

O

HN

OS

HN NH

OOH

Figure 10. Chemical Structure of the α-Chymotrypsin substrate.

Assays were started with low substrate concentrations of 2 µM and an α-chymotrypsin

concentration of 200 nM. Streptavidin agarose bead suspension (1 ml has a capacity of

≥ 85 nmol biotin) was used in the assays after optimization to determine the necessary

amount to pull down all the substrate (Figure 11).

26

Figure 11. Optimization of the streptavidin beads suspension used for assays. 1 µM of the

substrate was diluted 10 times and different amounts of streptavidin agarose bead suspension was added and reacted for 30 minutes at 25º C with shaking. The supernatant was analyzed by

ICP-MS.

An initial assay at low substrate concentrations of 2 µM and 200 nM α-chymotrypsin , an

81% conversion of the substrate was observed after about 120 minutes, calculated by

considering the positive control as a 100% activity of the enzyme where no biotin pull

down was used.

Unfortunately these results were not reproducible and investigations with higher

substrate concentrations showed less than 10% of substrate cleavage (Figure 12).

Figure 12. Solution phase ICP-MS α-chymotrypsin assay. Substrate concentration was 10 µM

(blue, ♦) and 2 µM (pink, ■) Enzyme concentration was 200 nM. The blank reading was subtracted from the samples.

00.20.40.60.8

11.2

0 µl 12 µl 15 µl 25 µl 30 µl 60 µl 80 µl 100µl

buffer

Amount of streptavidin agarose bead suspension

subs

trat

e co

ncen

trat

ion

(uM

)

0

200

400

600

800

1000

0 5 10 15 20 25 30

time (mins)

prod

uct c

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(nM

)

27

The reason for this was not completely clear. Assays with different α-chymotrypsin

concentrations at various time points (4 minutes, 15 minutes, 30 minutes, 26 hrs) were

run in order to observe a linear response between enzyme concentration and the

activity. However, a linear dependence on enzyme concentration was not observed. In

fact there was little difference in the velocity of the reaction when the enzyme

concentrations were increased by 10 fold or 100 fold or even a 1000 fold, and this result

was obtained repeatedly (Figure 13).

Figure 13. α-Chymotrypsin assay with different concentrations of enzyme. Substrate

concentration was 10 µM and α-chymotrypsin concentrations were 2 pM, 20 pM, 200 pM, 2000 pM and 20000 pM. The reaction was incubated for 15 minutes (green, ▪) and 30minutes (orange, ♦). Log of enzyme concentration versus product concentration (µM) plot is shown; the blank

readings were subtracted from the sample readings We postulated a few possible reasons for these observations :1) non specific

interactions of the product with the agarose beads 2) the product formed in the reaction

may be inhibiting the activity of the enzyme, or 3) inhibition of proteolysis by the trace

free lanthanides that may be present in the substrate stock43.

Experiments were set up to investigate these predictions. To test for non specific

interactions with the agarose beads a single turnover assay was run (Figure 14). This

means that there is an excess of the enzyme over the substrate which would ensure a

complete conversion of substrate to product. The substrate concentration used was 10

µM and the enzyme concentration was 20 µM. If there was any non specific interaction

0

50

100

150

200

250

300

350

400

450

0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5

log (enzyme concentration pM)

Pro

duct

con

cent

ratio

n (n

M)

28

of the product with the beads, the amount of cleaved product obtained would be less

than expected.

Figure 14. α-Chymotrypsin assay under Single turnover conditions. Substrate concentration was 10 µM and enzyme concentration was 20 µM.The reaction was carried out for 15 minutes. The

blank control contained the substrate without any enzyme and the positive control containined the substrate which was not treated with streptavidin agarose.

Since there was an 88% conversion of the substrate to the product it was concluded that

the non specific interaction of the product with the agorase beads was negligible (Figure

14).

To test whether traces of lutetium were inhibiting proteolysis an assay in the presence of

1 mM EDTA was run. EDTA is a metal chelator and would function to chelate any

unconjugated lanthanide contamination in the assay solution. It was observed that upon

heating the samples to stop the reaction, the EDTA pulled out significant amounts of

lutetium from the DTPA complex on the peptide substrate. Stopping the proteolysis by

lowering the pH to 2 had the same effect. Thus, 8 M urea which is a commonly used

protein denaturerant, was used to stop the reaction44. It is also know that streptavidin is

resistant to denaturation by 8 M urea45. However, urea and biotin have some structural

similarity and it can be envisioned that urea may also be able to inhibit biotin binding. To

test the proper binding of streptavidin to biotin on the substrate, a simple pull down

experiment was run in 8 M Urea (Figure 15).

0

1

2

3

4

5

6

7

8

20 µM α-chymotrypsin positive control blank control

conc

entra

tion

(uM

)

29

Figure15. Streptavidin pull down experiment in 8 M urea. Substrate concentration was 10 µM. Sample was diluted in 20 times in water or 8 M urea (5 µl in 100 µl). The substrate was pulled

down by streptavidin agaorose beads (70 µl, for 30 minutes at 25º C with shaking). The positive control contained the substrate which was not treated with streptavidin agarose.

The samples that were not treated with streptavidin agarose (positive control) were 15-

18 times higher in signal than the treated (blank control) sample. The graph clearly

showed that the substrate can be pulled down effectively using the streptavidin agarose

beads in 8 M urea under same reaction conditions. An assay in the presence of 1 mM

EDTA was run (Figure 16) using 8 M urea to stop the reaction, the DTPA-Lu complex of

the substrate was stable under this condition.

Figure16. α-Chymotrypsin assay in the presence of EDTA. Substrate concentration was 10 µM and enzyme concentration was 200 nM. The reaction was incubated for 26 hours. The blank

control contained the substrate without any enzyme and the positive control contained the substrate which was not treated with streptavidin agarose.

0

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300000

water 8 M urea pH 7.2

cps

(Lu) Blank control

Positive control

0

1

2

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4

5

6

7

NO EDTA EDTA (1 mM)

conc

entr

atio

n (u

M)

Blank control

200 nM enzyme

positive control

30

The result showed that the presence of EDTA, in fact did not increase enzyme activity.

This suggests that there was no significant inhibition of proteolysis in presence of the

trace lutetium.

To test for product inhibition an assay in presence of the product was performed. To do

this a single turnover assay was run for 15 minutes to generate product. The excess

enzyme was separated using spin columns and the flow through that contained the

product, was added to a multiple turnover assay (Figure 17)

Figure 17. α-Chymotrypsin assay in the presence of product. The substrate concentration was 10 µM and the enzyme concentration was 200 nM. The product formed after 2 hrs (orange) and the

product formed after 26 hrs (blue) are shown.

2.4 µM and 7.7 µM of product was formed after 2 hrs and 27 hrs respectively when no

external product was added. 1 µM and 5.2 µM of product was formed after 2 hrs and 27

hrs respectively in presence of 3 µM external product added. From the result obtained,

it was concluded that product inhibition, was not significant enough to explain the

enzyme inhibition observed at low enzyme concentrations.

To confirm the activity of the enzyme a synthetic fluorogenic substrate for α-

chymotrypsin (Suc-AAPF-AMC) 46 was purchased. This would allow for a direct

comparison of a fluorescence assay and the ICP-MS assay under development.

0

1

2

3

4

5

6

7

8

9

no Product added Product added (3 µM)

prod

uct c

once

ntra

tion

(uM

)

2hrs26hrs

31

4.2.4 Fluorescence assay using Suc-AAPF-AMC

A chymotrypsin fluorogenic substrate, Suc-AAPF-AMC was purchased to confirm the

activity of the enzyme stock. 20 µM or 5 µM substrate concentration in the reaction

buffer (100 mM tris pH 8, 10 mM CaCl2, 50 mM NaCl) and the appropriate enzyme

concentration was incubated at 25º C. The assay was essentially run like the ICP-MS

based assay with the elimination of the streptavidin agarose pull down step. Aliquots of

10 µl were diluted in 8 M urea (30 times diluted, 10 µl in 300 µl). This stopped the

reaction as well as brought down concentrations to detectable values. Blank samples

were run without the enzyme; it was assumed that a sample incubated with the enzyme

overnight represented a 100% conversion of the substrate to the product, which served

as a positive control. 100 µl of each sample was added to wells of a 96 well plate which

was read using a fluorescence plate reader. Fluorescence intensity was measured by

exciting the fluorophore at 380 nm and monitoring emission at 460 nm47.

A complete conversion of the substrate was observed within 30 minutes when 20 nM

enzyme concentration was used (Figure 18C) and about 5 minutes when 200 nM

enzyme concentration was used (Figure 18A, 18B). An assay with 2 nM, 4 nM, 8 nM, 20

nM and 200 nM enzyme concentrations was run (Figure 18D) and a linear response

between α- chymotrypsin concentration and product formation was seen.

32

Figure 18. α-Chymotrypsin Assay using Suc-AAPF-AMC as a substrate. A)-Substrate concentration was 20 µM. Enzyme concentrations were 20 nM (purple), 200 nM (pink). A blank control containing no enzyme was run (green). B) - Substrate concentration was 20µM. Enzyme

concentration was 200 nM. The assay was run twice, and the blank sample reading was subtracted from the test readings. C)-Substrate concentration used was 5 µM. Enzyme

concentration was 20 nM and the blank sample reading was subtracted from the test readings. D) Substrate concentration was 20 µM. Enzyme concentrations were 2 nM, 4 nM, 8 nM and 20 nM. A sample containing no enzyme served as a blank control and was subtracted from all the test

readings.

33

In conclusion, α-chymotrypsin assays with the fluorescent substrate, Suc-AAPF-AMC

showed a reproducible and complete turnover of the substrate. The enzyme activity was

clearly dependent on enzyme concentration and product formed was in linear

relationship with the enzyme concentration. This showed that the protocols used for the

α-chymotrypsin assay were acceptable and that the enzyme behaved as expected.

34

4.3 Calpain-1 assays Since experiments with α-chymotrypsin showed that the enzyme was not completely

turning over the substrate it was decided to change the enzyme and substrate pair. For

comparison calpain-1, a calcium dependent cysteine protease was chosen for the next

set of experiments. Fluorescent assays with a purchased synthetic substrate and ICP-

MS based assays with the terbium-tagged substrate that was synthesized were run in

parallel.

4.3.1 FRET assay

A synthetic FRET substrate specific for calpain-1, H-Lys(FAM)-Glu-Val-Tyr-Gly-Met-Met-

Lys(DABCYL)-OH was purchased to serve as a control and to check for activity of the

enzyme.

A substrate concentration of 10 µM was used since the Km is known to be 4.6 µM1. To

stop the reaction 8 M urea was used just as was done for the α-chymotrypsin assays. It

is a FRET substrate that is internally quenched and after hydrolysis by the enzyme the

fluorescence increases. The plot of the time vs fluorescence intensity was linear for

about 30 minutes when 200 nM enzyme concentration was added to the substrate and

for about 12 minutes when 500 nM enzyme concentration was used (Figure 19A, 19B).

From the slopes of the linear sections of the plots it can be seen that the rate of the

reaction seemed to co relate with the enzyme concentration.

35

Figure 19. FRET assay of calpain-1 using H-Lys(FAM)-Glu-Val-Tyr-Gly-Met-Met-Lys(DABCYL)-OH as a substrate. A)-Substrate concentration was 10 µM. Enzyme concentration was 200 nM. B)- Substrate concentration was 10 µM. Enzyme concentration was 500 nM.

An assay with different concentrations of calpain-1, 4 nM, 8 nM, 16 nM and 200 nM was

run for 10 minutes and 1 hour (Figure 20).

36

Figure 20. FRET assay using different concentrations of calpain-1. Substrate concentration was

10 µM and calpain-1 concentrations were 4 nM, 8 nM, 16 nM and 200 nM.The reaction was incubated for 10 minutes (blue) and 1 hour (pink). A)- Concentration versus fluorescence intensity

plot showing 0 nM,4 nM,8 nM and 16 nM concentrations of calpain-1. B)- Concentration versus fluorescence intensity plot showing 0 nM,4 nM, 8 nM, 16 nM and 200 nM concentrations of

calpain-1.

A linear relationship between the enzyme concentration and the fluorescence intensity

was observed after 10 minutes and 1 hour.

4.3.2 ICP-MS assay

Given that we saw good results with the FRET substrate, we went onto trying the

calpain-1 assay using the Tb-tagged substrate that was synthesized [DTPA- Glu-Val-

Tyr-Gly-Nle-Nle- Lys(biotin)] . The assay was run in a similar manner to the α-

chymotrypsin assay. The samples were diluted 40 times, to bring the levels of metal

within the range of detection.

Reaction conditions including buffers, temperature and concentrations remained the

same as the FRET assay. The streptavidin agarose was added and the detection of the

product was measured by ICP-MS. Thus, increase in the terbium counts would account

for increase in enzyme activity. The positive control was a sample containing no enzyme

and was not treated with streptavidin agarose which represents the maximum terbium

signal that could be observed.

37

Figure 21.A comparison of a FRET assay to an ICP-MS assay for calpain-1. The substrate

concentration is 10 µM and the enzyme concentration was 1 µM. The assay was run for 4 hrs at 25º C in the calpain-1 reaction buffer.A) FRET assay, B)ICP-MS assay

The shapes of the plots obtained for the FRET and the ICP-MS assays were very

comparable (Figure 21) .For the ICP-MS assay the plot started to plateau at about 60

minutes where the product formed was about 0.6 µM. After 180 minutes too, there is

only about 0.6 µM of product. The positive control for the assay showed Tb counts of

568927 cps after a 100 times dilution, which worked out to a concentration of 8.7 µM.

Thus, there was less than 10% conversion that was observed at the plateau of the plot.

The enzyme did not turnover the substrate efficiently and failed to produce the expected

amounts of product.

An assay similar to the FRET assay with different concentrations of calpain-1 was tried

with the Tb-tagged substrate. But there was no linear relationship observed between the

38

enzyme concentration and the product formed, in fact there was only a very small

difference in the terbium signal as the enzyme concentration was increased (Figure 22)

Figure 22. ICP-MS assay using different concentrations of calpain-1. Substrate concentration was 10 µM and calpain-1 concentrations were 20 nM, 60 nM 120 nM and 240 nM.The reaction was incubated for 10 minutes (blue, ♦) and 1 hour (pink, ▪). To confirm that the Tb-tagged substrate was being cleaved by calpain-1 an assay under

single turnover conditions was run. The substrate concentration used was 5 µM and

calpain-1 concentration was 5 µM. This would ensure a complete conversion of the

substrate to the product (Figure 23).

Figure 23. Calpain-1 assay under single turnover conditions. The substrate concentration was 5 µM and enzyme concentration was 5 µM. A) The substrate was incubated with the enzyme for

45minutes B) The substrate was incubated with the enzyme 2 days.

0.35

0.45

0.55

0.65

0.75

0.85

0.95

0 50 100 150 200 250

Calpain-1 concentration (nM)

Pro

duct

con

cent

ratio

n (u

M)

39

Calpain-1 showed a 25% conversion of the substrate to product in 45 minutes and a

95% conversion in 2 days. This assay showed that the sequence synthesized was

cleaved by calpain-1 when large concentrations of enzyme were used.

Calpain-1 assays with the FRET substrate, H-Lys(FAM)-Glu-Val-Tyr-Gly-Met-Met-

Lys(DABCYL)-OH showed good conversion of substrate to product under multiple

turnover conditions and there was a linear relationship between enzyme concentration

and product formed. However, less than 10% of the Tb-tagged substrate was converted

to the product under the same conditions and there was no linear relationship observed

for the product formed as calpain-1 concentration was increased.

Similar behaviour was seen with both α-chymotrypsin and calpain-1 ICP-MS assays.

Both enzymes were able to turnover control fluorogenic substrates effectively but with

the Ln-tagged substrates the proteases seemed to be inhibited and failed to produce the

product that was expected. The previous observation that nearly complete turnover of

the Lu-tagged α-chymotrypsin substrate can be achieved at long timepoints (Figure

16,17) suggest that the substrate binds tightly and is cleaved very slowly.

4.3.3 Competition assay of Calpain-1

The observations from the ICP-MS assays showed that calpain-1 was unable to turnover

the Tb-tagged substrate and the expected amount of product failed to be formed. To

investigate this further, a competition assay was run. A fluorescence assay for calpain-1

was run in presence of the Tb-tagged substrate. This was followed by an ICP-MS

analysis of the samples to verify for the cleavage of the Tb-substrate added.

The total concentration of substrate was 10 µM. The FRET substrate and Tb-tagged

substrate were incubated with the enzyme in two different ratios 5 µM: 5 µM and 7.5 µM:

2.5 µM respectively. An assay with 5 µM of FRET substrate without any Tb-tagged

40

substrate was also incubated with the enzyme for comparison. The calpain-1

concentration used was 1 µM and the assay was run for 120 minutes (Figure 24).

Figure 24: Competition assay for calpain-1. Substrate concentrations were 5 µM FRET substrate (red, ♦), 7.5 µM FRET substrate and 2.5 µM Tb-tagged substrate (blue, ▲). 5 µM FRET substrate

and 5 µM Tb-tagged substrate (green, ▪). Enzyme concentration was 1 µM.

When 5 µM of the FRET substrate was incubated with the enzyme and no Tb-tagged

substrate was added the plot starts to plateau at about 30 minutes. When 5 µM of the

Tb-tagged substrate was added the fluorescence intensity was reduced to half at every

time point and product formation stopped after 15 minutes. In the presence of 2.5 µM of

Tb-tagged substrate a similar effect was seen, where at early time points upto 15

minutes the signal was higher due to presence of more FRET substrate as expected, but

after 15 minutes more product failed to be formed. From the ICP-MS analysis of the

samples it was seen that only 1.6% of the 5 µM Tb-tagged substrate was cleaved after

120 minutes and a negligible amount of 0.02% of the 2.5 µM Tb-tagged substrate was

cleaved after 120 minutes. This indicates that the Tb-substrate was behaving more like

an irreversible inhibitor than a competitive inhibitor.

Results seen with the competition assay experiment suggest that calpain-1 was being

inhibited, in an irreversible manner by the Tb-tagged substrate since more fluorescent

product failed to form after 15 minutes in presence of either 2.5 µM or 5 µM of Tb-tagged

substrate.

0

10000

20000

30000

40000

50000

60000

0 20 40 60 80 100 120 140

time (min)

Fluo

resc

ence

inte

nsity

41

The Tb-tagged substrate contains the DTPA ligand chelated to the terbium at the N-

terminus. A solution to the observed inhibition may be to increase the distance between

the peptide sequence of interest and the DTPA ligand which may prevent the rather

bulky DTPA complex from interacting with the active site of the enzymes. Further studies

into the mode of inhibition of the DTPA tagged substrates are ongoing.

42

5 Conclusion and future work

Here we report the design and synthesis of lanthanide-tagged protease substrates for

the development of an ICP-MS based protease assay.

Our results showed that DTPA could be easily coupled to peptide substrates, which

were then successfully and quantitatively tagged with lanthanide metals. The DTPA-Ln

complex was stable and there was no observed release of the metal from the complex

during the enzyme assays under the reaction conditions used.

Promising results were seen with the initial α-chymotrypsin and calpain-1 assays. A

complete turnover for the lanthanide-tagged substrates was observed under single

turnover assay conditions. The general low turnover of the substrates under multiple

turnover conditions as well as the results from the competition calpain-1 assay suggests

that the lanthanide-tagged substrates were inhibiting proteolysis.

Since assays with the control fluorogenic substrates for α-chymotrypsin and calpain-1

displayed expected activity, it led us to believe that the presence of the DTPA-Ln

complex has a role to play in the inhibition of the proteolysis that was observed in the

assays using the lanthanide-tagged substrates. Increasing the distance between the

rather bulky DTPA-Ln complex and the peptide sequence of interest might prevent the

metal complex from interacting with the active site of the enzyme and thus overcome

any inhibition of proteolysis due to the presence of the metal complex. This could be

achieved by the addition of a spacer between the peptide substrate sequence and the

DTPA-Ln complex. It has been shown that a spacer comprised of four β-alanine

residues can be effective at achieving a significant separation31.Since β-alanine does

not occur in nature, it would not be recognized by the proteases and would serve as a

spacer without interfering in the enzyme assays. Using β-amino acids also provides a

longer spacer in comparison to using α-amino acids.

43

Although we have used α-chymotrypsin and calpain-1 to develop and illustrate this

analytical technique, many other proteases like the caspases, MMPs and ADAMs can

be detected in a single assay or in a multiplexed fashion. Given the great sensitivity and

excellent spectral resolution of the ICP-MS system, we can develop a quantitative and

reproducible assay.

The future direction would be to develop a multiplexed assay for proteases using

multiple orthogonal substrates. This could be done on an encoded solid support using

TentaGel beads or in solution phase using biotinylated protease substrates.

44

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7 Appendices 7.1 HPLC Traces

7.1.1 α-Chymotrypsin substrate containing the fluorophore

7.1.2 α-Chymotrypsin substrate containing a DTPA tag and a biotin label

49

7.1.3 MMP-9 substrate containing a DTPA tag and biotin label

7.1.4 Caspase-3 substrate containing a DTPA tag and a biotin label

7.1.5 Calpain-1 substrate containing a DTPA tag and a biotin label