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2 Protein Arrays, a Variety of Detection Approaches

2 Protein Arrays, a Variety of Detection Approaches

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Chapter 9



MICROARRAYS AND DYNAMICS OF FLUORESCENT DYES



of techniques implicates numerous applications for

proteomics studies, including biomarker discovery,

protein interaction studies, enzymeesubstrate

profiling, immunological profiling, and vaccine

development, among many others. The need to

detect extremely low-abundance proteins in complex

mixtures has forced the development of sensitive,

real-time, and multiplexed detection platforms

shifting the main research effort from academia to

biotech industry. Several protein microarray systems

are now available commercially, including ProteOnÔ

(Bio-Rad Laboratories), ProtoArray (Invitrogen), or

various products from Arrayit “R” Corp.

Protein microarrays are made by immobilization of

a number of purified proteins, protein domains, or

functional peptides and are generally used to study

proteineprotein interactions for screening purposes.6

However, protein-detecting microarrays are made by

the immobilization of specific protein capture

reagents, which are any chemical species that interact

selectively with the target protein.7,8 These microarrays

are used for protein profiling, that is, quantification of

protein abundances and evaluation of post-translational modifications in complex mixtures.9

Several methods of immobilization have been

proposed up to date. It is worth mentioning chemical

immobilization, including click chemistry methods,10

His tag trapping,11 and cysteine trapping on gold

surface,12 as well as physical immobilizations such as

biotin streptadivin interaction,13 simple adsorption,

hydrophobic or electrostatic,14 hydrogen bonding,

and so on. Another approach is to synthesize proteins

on a chip using in situ synthesis where arrays are

applied on the chip as mRNA strands, and further

using the transcription machinery (ribosome

enzymes and nucleotide mixture), a target protein is

synthesized.15

After successful attachment of protein or peptide

on the chip, followed by sample application, detection of the positive/negative interactions that took

place on the chip may be accomplished using one of

several systems. The first type of detection system

requires an additional molecule that could be easily

recognizable and detectable by the microarray scanning system. Conventional label-based approaches



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tag the target molecule with fluorescent dyes and

radioisotopes16,17 or, more recently, tags in the form

of inorganic quantum dots (Qdots). A second method

used to analyze the protein chip is a nonlabeled

approach of detection; it usually requires state-ofthe-art instrumentation, such as high resolution mass

spectrometry (matrix-assisted laser desorption/ionization, MALDI), which allows direct ionization from

the chip surface and further identification of the

generated ions.18 Another system that follows the

interaction between immobilized (in this case on the

gold surface) protein or ligands is surface plasmon

resonance. The output of this technique is to examine

changes in the reflection angle of light that hits the

gold surface in which upon formation of proteineligand complex the reflection angle is changing

proportional to the strength of such binding.



9.3 Fluorescence Labeling

9.3.1 Introduction of the Fluorophore

Among all the mentioned techniques of monitoring interactions on the chip, both qualitatively and

quantitatively, the most common way is fluorescent

based. In most cases, fluorescent chemical moieties

are needed to label the protein or peptide before, or

usually more often after, the target molecules have

been attached to the slide. Multiple approaches yield

a fluorescent biomolecule(s): introduction of the large

protein via the biosynthetic pathway, modification of

the functional group in the protein sequence by small

chemical moiety with fluorescent properties, or

introduction of amino acid fluorescent moieties. The

first is to fuse a target protein with fluorescent protein

[green fluorescent protein (GFP) and yellow fluorescent protein (YFP)]; these are especially useful for

labeling proteins obtained by gene expression inside

cells.19e21 This approach is usually used for large

proteins resistant to structural changes resulting from

the introduction of GFP/YFP (both 27-kDa proteins).

Another class of labeling reagents is low molecular

weight organic fluorescent dyes.22,23 In general, the

organic fluorescent moieties are modified selectively

through a functional group (NH2-, COOH-, HS- and



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MICROARRAYS AND DYNAMICS OF FLUORESCENT DYES



OH-, etc.) in the proteins, peptides, and nucleic acids.

To enhance reactivity with functional groups, the

organic fluorescent dyes are activated by introducing

reactive chemical moieties, such as N-hydroxysuccinimidyl ester, maleimide, and halogens. Alternatively, relatively short peptides and nucleic acids

can be modified directly using fluorophores by solidphase peptide synthesis.

Many non-natural amino acids containing organic

fluorescent dyes in the side chain have been

developed to date (fluorescent amino acids;

R



R=



Fmoc



R



O



HN



H

N



COOH



HN

R







Ant

(359/407)



Edn

(344/468)



O



NH

O

O O



NH

O

O O HO



O



N

H

Bad

(354/512)



R

R



N



NH

O

O O HO



O NH



R

R

NH

O



NH

O

O



O O



O O



N



Coc

(350/423)



Hmc

(327/393)



Fam

(501/524)



Dec

(420/465)



NH

NH

O



O



HN O



O



O



N



R

R



COOH

N



O



O O



Mac

(377/464)



R



NH



COOH

HO



NH

O

O O



O



R

NH

O



NH

O

O O



Cm3

(446/491)



R



O O

Cmr

(325/380)



Pyr

(344/379)



R



R



Hoc

(411/451)



Mca

(336/378)



Moc

(348/400)



O



O

Aca

(354/444)



R

R



R



O

R



N

N

H

Acd

(401/421)



SO3H



R



NH

O



O

R



N



O

Tmr

(542/569)



N+



N

H



O

@52

(525/545)



N+

H



HO3S



O O

CF3



A43

(437/534)



+



N



O

O

N

S39

(389/528)



Figure 9.1 Chemical structures of fluorescent amino acids. Maximum excitation/emission

wavelength is shown in parentheses. The Edn is a g-[2-(5-sulfonaphtalen-1-ylamino)ethyl]

amide-L-glutamic acid; Ant, b-(2-anthryl)-L-alanine; Acd, b-[acridine-9(10H)-on-2-yl]-L-alanine;

Aca, N-d-(9-oxoacridin-10(9H)-acetyl)-L-ornithine; Bad, b-[benzo[b]acridin-12(5H)-on-2-yl]-Lalanine; Pyr, b-(1-pyrenyl)-L-alanine; Cmr, b-[4-(7-methoxycoumaryl)]-L-alanine; Moc, N-ε-(7methoxycoumarin-4-carbonyl)-L-lysine; Mca, N-ε-(7-methoxycoumarin-4-acetyl)-L-lysine; Hoc,

N-ε-(7-hydroxycoumarine-4-carbonyl)-L-lysine; Hmc, N-ε-(7-hydroxy-4-methyl-coumarin-3acetyl)-L-lysine; Coc, N-d-(6-oxo-6H-[1,3]dioxolo[4,5-g]chromen-8-acetyl)-L-ornithine; Mac,

N-ε-(7-dimethylaminocoumarin-4-acetyl)-L-lysine; Dec, N-ε-(7-diethylaminocoumarin-3carbonyl)-L-lysine; Cm3, N-d-(coumarin 343-3-carbonyl)-L-ornithine; Fam, N-ε-(fluorescein-5(6)carbonyl)-L-lysine; Tmr, N-ε-(tetramethylrhodamine-5(6)-carbonyl)-L-lysine; @52, N-d-{(Z)-N-(6(ethylamino)-2,7,dimethyl-3H-xanthen-3-ylidene-9-ethylcarbonyl)}-L-ornithine; A43, N-ε-{(8,8dimethyl-2-oxo-4-(trifluoromethyl)-8,9-dihydro-2H-pyrano[3,2-g]quinolin-6-yl)methanesulfonic

acid-9-pentylcarbonyl}-L-lysine; S39, N-ε-(4-(2-(chroman-6-yl)oxazol-5-yl)-1-benzylpyridinium-3carbonyl)-L-lysine.23e25



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Figure 9.1).24,25 Modified amino acids can be incorporated directly with arbitrary positions of the peptide

chain, yielding labelled peptides, further utilized to

investigate its activity or interactions with target

molecules (i.e. receptor, enzyme etc.)26. If the fluorescent amino acids are site-specific incorporated in

proteins, it is also possible to investigate the activity of

these proteins and interactions with molecular

targets.



9.3.2 Examples of Fluorescent Dyes and

Application to Biological Systems

The first labeling method for the detection of

biomolecules was the use of radioisotope entities

such as 32P, which have been employed successfully to study proteineprotein, proteineDNA,

protein eRNA, and proteineligand interactions.

More recently, however, radioisotope utilization as

labels has declined due to safety and health

concerns.27 A fluorophore as a suitable alternative

has been developed and widely accepted. Dyes

such as rhodamine, fluoroscein, phycobiliproteins,

nitrobenzoxadiazole, acridines, boron-dipyrromethene (BODIPY), and cyanine compounds or their

derivates are used most commonly for protein

microarray detection. Examples of commonly used

fluorescent dyes are presented in Figure 9.2. The

choice of fluorophore molecule to be used for

detection depends on the sample type, substrate,

number of proteins in the experiments, and light

emission spectra characteristics.28 Cyanine dyes,

Cy3 and Cy5 (Figure 9.2A), are good examples of

dyes used in microarray detection due to their

brightness and ability to label proteins easily with

the ε-amino group of lysine residues.29 Detection

using fluorescence labeling can be performed in

two ways: direct labeling (one antibody assay) or

indirect labeling.30 Using the direct labeling

method, the selected protein is labeled with a fluorophore (Cy3 or Cy5), which binds to antibodies

immobilized on the surface of the chip. This

method allows simultaneous incubation of a reference sample with the experimental sample, both



Chapter 9



MICROARRAYS AND DYNAMICS OF FLUORESCENT DYES



(A)

N

+



R



N

R

Cy3



N





+



N

R



R

Cy5



(B)



(C)

N



O



OH



O



+



_



N



B

F



F



BODIPY FL



HOOC

COOH



FITC

N

C



H2N

N



O



S

CI· +

NH2



+



_



N



B

F



F



HN



BODIPY 650/665



TRITC

O

COOH



Figure 9.2 Chemical structure of commonly used fluorescent dyes from (A) cyanine, (B)

rhodamine, and (C) BODIPY families.



having different dyes attached.31 However, it is

worth noting that this approach allows detecting

relatively highly concentrated proteins. A dual

fluorescence method has been used for the detection of biomarkers in prostate cancer. Serum



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samples from 33 patients and 20 healthy controls

were analyzed on a microarray immobilized with

180 antibodies and revealed that nine proteins were

found unique to prostate cancer patients.31 Detection limits of 6.25 pg of the protein have been

achieved successfully using Cy dyes.32

BODIPY (Figure 9.2C) is a highly fluorescent

molecule alternative for Cy dyes. Pei and coworkers33 constructed a chip that was used for performing assays of G-protein GTPase activity using

BODIPY-GTP as the enzyme substrate. Active protein

G releases the highly fluorescent BODIPY moiety

from the BODIPY-GTP molecule. Such a chip is used

for screening modulators (inhibitors or activators) of

the G protein. The enzymatic activity was measured

by assaying the amount of fluorescent product

formed in the enzyme reaction (BODIPY) mixtures

that contained test compounds.33

The BODIPY dye was utilized further to develop

and characterize the fluorimetric acetylcholinesterase (AChE) assay, both in solution and with the

enzyme entrapped in sol-gel-derived silica.34

Additionally, this assay was extended to the

manufacturing of functional AChE microarrays. It is

based on a disulfideethiol interchange reaction

between the intramolecularly quenched dimeric

dye, BODIPY FL L-cystine and thiocholine, generated

by the AChE-catalyzed hydrolysis of acetylthiocholine, which results in a brightly fluorescent monomeric product due to cleavage of the disulfidecoupled form of the dye. Sixty-six different colloidal

solutions (various solution-gel ratios) produced

robust microarrays; among them, 26 combinations

were identified that could produce highly active

AChE microarrays.35

In the indirect labeling method, target proteins

are captured by immobilized antibodies. Detection is

then performed by utilization of a fluorophorelabeled secondary antibody molecule. This technique is much more specific compared to the direct

labeling method, as positive binding requires

a simultaneous interaction between the protein and

two different antibodies directed to two different

epitopes. This results in a decreased fluorescent

background and increased sensitivity.36 One major



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MICROARRAYS AND DYNAMICS OF FLUORESCENT DYES



shortcoming is the limited availability of required

selective primary antibodies.

Light emission (luminescence) is another method

used to visualize the target/captured protein. Biotin

market proteins or proteins attached to a Strep tag

(peptide) that has a high affinity for streptavidin are

applied on the microarray system. A light intensity

boost is observed as positive output after incubation

with streptavidin conjugated with fluorophore37 or

horseradish peroxidase in the presence of an appropriate substrate.38 In the latter paper using this biotin

label-based antibody array technology, the authors

measured expression levels of 507 human, 308

mouse, and 90 rat target proteins can be simultaneously detected, including chemokines, growth

factors, cytokines angiogenic factors, proteases,

soluble receptors, soluble adhesion molecules, and

other proteins in a variety of samples. Most proteins

can be detected at picogram per milliliter and

nanogram per milliliter levels.

The problem that many research groups face in

dealing with protein arrays is proper and accurate

detection of the dynamic range of the protein amount.

This is an especially important issue when using

a labeling method of detection. Most fluorescent

groups or dyes are not efficient/sensitive enough to

ensure a proper readout for low abundant proteins.

A rolling circle (RCA)-based amplification protein

detection method, referred to as immuno-RCA, has

been developed39,40 as a superior method for signal

amplification in protein microarrays. This is due to the

isothermal amplification process, which preserves the

integrity of the antigeneantibody complexes and

maintains the spatial separation required for multiplexing on microarrays. RCA-based indirect immunoassays on microarrays involve the crucial steps of

sample protein captured by a specific antibody affixed

to a chip, followed by binding of a second biotinylated

detector antibody to captured proteins. Next comes

binding of a universal antibody to a secondary antibody and finally RCA signal amplification on the

universal antibody. RCA occurs predominantly on the

universal antibody via the covalent attachment of

oligonucleotide primers. Upon hybridization with

DNA circles, the DNA polymerase extends primers



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along the C probe, resulting in a considerably intensified signal. The amount of on-chip synthesized DNA

could be measured using intercalating dye that integrates with double-stranded DNA. Conversely, RCAbased direct immunoassays can be performed in

which RCA signal amplification takes place on an

analyte-specific secondary antibody. Using this

approach, one can enhance the protein detection

limits significantly.



9.3.4 Qdots

Qdots41,42 are another group of fluorescenceemitting molecules. The Qdot is a nanometal fluorophore with a bright and linear signal of emitted

light. One advantage of using a Qdot is that it has no

photobleaching effect, which often occurs while

using organic fluorophores. Combined with narrow

emission spectra and a greater quantum yield,43

Qdots make an excellent alternative for conventional

fluorophores. Indeed, Qdots find a diverse range of

applications in biological sciences, such as diagnostic imaging,44 detection of cancer biomarkers,45,46

and probing of human serum.47 However, the low

stability and short lifetime of Qdots due to their

susceptibility to oxidation are the main shortcomings

of these methods.

Another strategy important in evaluating protein

target interactions is fluorescence resonance energy

transfer (FRET).19,21 Because the efficiency of FRET

depends on the distance between target molecules

(containing donor and acceptor of fluorescence), this

effect is adapted easily for two labeled molecules.

Single molecules display high fluorescence intensity,

and the detection of proteineprotein binding on

microarrays using the fluorescence lifetime was

described by Nagl and co-workers.48 The authors

introduced efficient FRET donor/acceptor pair (dyes

Alexa 555 and Alexa 647) in a competitive assay

format on three different microarray surfaces. Lifetime maps were recorded, and interaction between

the proteins could be detected clearly on all formats

and resulted in almost complete quenching on the

slide surface upon addition of excess streptavidin

labeled the FRET acceptor dye. The method could be



Chapter 9



MICROARRAYS AND DYNAMICS OF FLUORESCENT DYES



used to assess all types of protein interaction analyses on microarrays.

Another important application is to follow the

activity of proteolytical enzymes by FRET substrates.

This issue is described excellently in the work of

Dı´az-Mocho´n and co-workers49 where they synthesized and analyzed 10,000 members’ peptide nucleic

acid-encoded library of FRET-based peptides for

global analysis of protease cleavage specificity.

Analysis was achieved using a microarray and

consumed minimal quantities of enzyme (60 pmol)

and library (3.5 nmol).



9.4 Closing Remarks

The protein microarray platform is a dynamic and

expanding area of proteomic science that allows high

throughput analysis. The progress of hardware

“shrinking” (nanoarrays are on the horizon), correlated with a reduction of the amount of required

analyte, is a serious challenge. There is a need to

improve the final readout in the sense of the sensitivity and stability of the fluorophores. Novel fluorescent materials such as Qdots are proven progress

in this area. Luminescence-based detection or its

combination with a fluorescent one is a good

example of upcoming systems. Other nonconventional methods, such as rolling circle amplification,

ensure the ability to detect as little as a nano- or

subnanomolar range of the protein. A label-free

detection method in the near future will become

a serious alternative to label-based detection and

science will benefit from such competition.



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