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BME1450, Nov 2004
1
Protein Microarrays: Quantitative, Temporal
Proteomic Data for Systems Biology
Alborz Mahdavi

Abstract: Considerable advances have been made in the area of
cell signaling models, but in order for these models to be
biologically relevant, precise, time dependant, quantitative and
high-throughput proteomic data is required. Several methods to
detect proteins such as non-isotopic labeling based techniques,
quantum dots, mass spectrometry, surface plasmon resonance and
colourimetric resonant reflection are considered. The principle
behind each technique as well as its limitations is discussed.
Utilization of surface plasmon resonance in conjunction with selfassembling, antibody-free probes is proposed as a platform for
protein microarrays. It is further suggested that this system can
be used for temporal analysis of protein phosphorylation events
as well as determination of protein binding constants; two key
pieces of information that are essential for the development of
useful cell signaling models for systems biology.
Index Terms—Microarrays, Surface Plasmon Resonance, Selfassembled, Antibody-free, Proteomic, Systems Biology
I. INTRODUCTION
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mportance of quantitative protein detection is one of the
realizations of the post-genomic era. Predictions of such cell
signaling models as Bayesian networks, differentiation
equation based models, Boolean networks and protein
networks are only as good as the data that is used to generate
the model. Currently technological restrictions limit these
modelling approaches because the quantification of protein
changes such as phosphorylation and dissociation rates are
cumbersome and imprecise. Hence development of accurate
protein detection technologies is needed. Protein detection will
be useful not only for determination of signaling events in
cells, but it has applications in food engineering,
environmental sampling, quality control, bio-agent detection as
well as screening for diseases such as HIV and Hepatitis.
Solving this problem will not only benefit research, it will have
immediate impact in industry. Two criteria have to be met for
any protein detection technology to be effective. First, the
protein must be physical tagged using a molecular marker, and
second the protein-probe complex must be detected with high
precision. These two steps may be combined, however it has to
be feasible to make the system high-throughput so that a large
number of samples are screened for a large number of different
proteins. In order to consider the problems associated with
protein detection, it is informative to reflect on how DNA
microarrays circumvent similar problems.
II. DIFFICULTIES IN PROTEIN DETECTION
A. DNA Microarrays
DNA microarrays consist of a surface onto which DNA is
synthesized using photolithography techniques. Four masks
corresponding to nucleotides AGTC are used in
photolithography to make probe strands of about 25
nucleotides; a total of more than 500 000 probe locations can
be made on one array [11]. Since the probes are made in
parallel, each probe spot will contain many identical single
strands of DNA. The mRNA sample that is to be analyzed is
first amplified in a process called polymerase chain reaction
(PCR) using florescent nucleotides, which replicates the DNA
strands many times so that a high concentration of sample
cDNA is available [7]. This sample cDNA is then washed over
the surface of the microarray and complementary strands
attach. Since florescent nucleotides were used in the sample,
the matching pairs can be detected using laser [11]. For a more
detailed explanation of the techniques refer to [11]. Several
factors contributed to the success of DNA micro arrays. The
photolithography instruments were already available from
electronics manufacturers, the sample could be easily
amplified by PCR, the amplified sample was already
fluorescent and would bind specifically to its complementary
strand. The problem with protein microarrays is that each step
of this process has to either be modified or resolved.
B. Problem of Tagging Proteins
Robust tagging of proteins is required for immobilization of
protein of interest at the predefined spot on the array for
detection. Antibodies are the best example of highly specific
protein tags [7]. The specificity of antibodies and their high
binding constant (affinity for their protein) makes them ideal
for detection of proteins, provided that the antibody is
available [12]. However, it is not cost-effective to purchase
100000 antibodies for a typical protein microarray.
Furthermore, if such an array is built, it will be very difficult to
store the array because the antibody proteins will denature
over time [5]. It is also difficult and time-consuming to
produce purified antibodies for proteins that are not
characterized and are newly found. Thus, what is needed is
essentially a replacement for natural antibodies. The tagging
agent must be highly specific, easy to produce on the array and
have the ability to attach to the surface so as to hold the
protein for detection. Furthermore the tags must be easily
stored and modular, such that many different tags are
available, each corresponding to a specific protein. DNA
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microarrays do not suffer to the same extend from this
problem, because the probes are already linked to surface and
highly specific for their complementary strand.
C. Problem of Labeling and Detection
Detection of the presence of a certain protein, even if it is
immobilized at the correct spot on the array is not easy. The
problem arises from the fact that if the original concentration
of the protein in the sample is very low, the detection of such
low concentration is very difficult. The equation governing the
amount of protein immobilized to the surface based on first
order kinetics is:
Ceq 
RT L0
L0  K D
for a different length of time. The cells are lysed, their contents
mixed in equal ratios, phosphorylated proteins separated, and
MS is performed on the sample. The results are as depicted in
figure 1. Using this approach, protein phosphorylation events
can be studied.
(1)
Where RT is the amount of tag on the surface, L0 is the
amount of protein in the sample and KD is dissociation
constant [9]. As L0 is often low, the amount of bound protein is
highly affected by differences in dissociation rate constant and
amount of tags present on the surface. For a constant surface
tag density; differences in binding affinity will dominate the
strength of detected signal and produce non-uniform noise
[12]. Furthermore, whereas in DNA microarrays the amplified
sample DNA strands are already fluorescent, in protein
microarrays the proteins are not fluorescent and hence another
highly specific tagging step is required to be able to detect the
proteins.
III. APPROACHES TO LABEL AND DETECT PROTEINS
A. Mass Spectrometry
Protein Mass Spectrometry (MS) involves charging proteins
in the presence of a very strong electric field. Depending on
the mass to charge ratio of the protein, time of arrival at the
electrode is different for different proteins as they travel
through the field [7]. Hence exact determination of proteins in
the sample is possible through MS. Furthermore proteolytic
fragmentation of the proteins allows for determination of the
constituents parts. The most recent and widely used MS
technique for protein detection is Matrix Assisted
Deposition/Ionization Mass Spectrometry (MALDI MS) [10].
In MALDI the protein is dissolved in low molar ratio in an UV
absorbing organic acid (the matrix), which is then vaporized
using a strong UV laser. UV laser transfers some of the
protons of the matrix to the protein and charges the protein
whilst vaporizing the matrix [10]. This approach is a softer
alternative to normal MS, and can accurately determine protein
constituents.
Recently MS has been used to perform temporal analysis of
phosphorylation events in cells [6]. Cell populations from
which peptides are extracted are first grown in medium
containing different isotopes incorporated into the amino acid
Arginine. For example, 13C14N and 12C14N and 13C15N
Arginine are used where the number depicts the mass number
of carbon or nitrogen [6]. Thereafter the cells are subjected to
different duration of signal activation through introduction of
cytokine or soluble growth factors. Hence each different
isotope combination contains a different amount and
composition of phosphorylated protein because it is stimulated
Fig1. Two different cell populations can be distinguished by a 4 Dalton
shift which results from presence of heaver isotopes in second cell medium.
The heavier isotope cell population is stimulated for 10 minutes and an up
regulation of phosphorylated peptides is observed from the 5-minute time
point from increase in relative intensity.
MS would be the strongest competitor of any protein
microarray technology due to its high-throughput nature;
however MS has its limits. Although MS can theoretically be
used to find all the proteins in a sample, sophisticated
purification through affinity columns is required. Cells must be
grown in defined mediums and samples from endogenous
tissues cannot be used in the above approach. Furthermore, use
of isotopes for temporal analysis limits the number of time
points that are possible. MS is highly statistical and often done
in conjunction with another protein separation technique
because presence of peaks in the MS spectra must often be
verified [10]. For brevity, these techniques will not be
discussed here and reader is urged to consult source [2] for
more details.
B. Non-isotopic Labeling and Quantum Dots
Non-isotopic labels can be categorized into four groups,
organic reporters that are enzyme activated, luminescent,
fluorescent and electro-active labels [7]. These labeling
techniques are based on emission of light, which is detected
using a photo-sensor. The amount of emitted light is correlated
with the amount of labeled protein, in the case of protein
detection. Fluorescent labels in particular are excited at one
wavelength and emit at a different wavelength. For example
Argon laser is used to activate the fluorescent dye FTIC at 488
nm, which emits light at about 530 nm. Depending on the
precision of the detecting instrument, currently about up to 8
different dyes can be detected at once. Quantum Dots (QD) are
ultra-sensitive fluorescent dyes that have three main
advantages over other fluorescent dyes. The dots size can be
used to tune the emission wavelength for about 20 different
colours, the excitation spectra is much wider and the emission
spectra is about one third as wide as normal fluorescent dyes
[4]. QDs are about 20 times brighter and 100 times more
stable [4]. However, even with the advent of QD technology,
there are significant problems associated with these labeling
systems for use in protein microarrays. The labels will still
have to be attached to the tagged proteins on the surface of the
array and cannot be incorporated into the protein. The number
BME1450, Nov 2004
of possible colours is limited for high-throughput detection,
and significant background noise is present. Furthermore,
although QD’s do not suffer from photo bleaching, other
fluorescent dyes do, and even QD’s are not useful for longterm continuous detection because protein denaturing becomes
a problem. As such more novel techniques must be used for
detection of proteins on the surface of the microarray.
C. Surface Plasmon Resonance
Surface Plasmon Resonance (SPR) sensors are optical
sensors that make use of changes in electromagnetic properties
of sensor surface for detection [3]. This is achieved by
producing a Surface Plasma Wave (SPW) at the interface
between a metal and a dielectric by shining a light that has the
same component wave vector as the SPW [3]. SPW
propagates along the interface and behaves like quasi-free
electron plasma, attenuating exponentially from the interface
and is extremely sensitive to the complex dielectric constant of
the medium and the interface [3]. The propagation constant for
SPW is solely based in the electric constant and the
wavelength of light that is used to induce it.
SPR sensors use a prism of high refractive index to achieve
a total internal reflection at the interface with a metal, giving
rise to an evanescent field wave, which penetrates the metal
surface into the medium containing the analyte [3]. Once the
evanescent field wave constant matches that of SPW, changes
in dielectric constant of the surface, for example by addition of
protein on the surface, will dramatically change the SPW.
SPW interacts with light so that changes in SPW can be
correlated with changes in spectral distribution, polarization,
amplitude and phase of reflected light. These can be detected
as a measure of presence of anlyte on the surface.
The detection method is indirect and label-free. The setup for a
SPR sensor is as follows:
Fig2. Schematic representation of the SPR optical sensor shows specific
attachment of protein to its corresponding tag and change in evanescent field
wave. Prism with a high refractive index is used to direct light.
There are several advantages to SPR sensors including realtime and continuous detection of proteins as well as the ability
to detect proteins without the need for labeling. Furthermore,
since the tags are not effected by detection, the sensor can be
used for extended periods of time and problems such as photo
bleaching are not encountered [3]. In cases where labeling of
proteins will alter their structure, SPR offers an alternative
detection technique. Real time detection of proteins using SPR
only became available recently, and advances in micro-fluidics
and photonics have paved the way for progress of this
technology.
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D. Colourimetric Resonant Reflection
Colourimetric Resonant Reflection (CRR) makes use of
changes in the refractive index of surfaces caused by presence
of analyte as a method of optical detection [1]. Although CRR
is based on similar principles as SPR, the sensor setup and
capabilities are different. In CRR light passes through the
analyte solution and hits a specially designed grating at the
bottom of the surface that reflects only a single frequency [1].
Presence of proteins on the surface causes a change in the
refractive index of the surface. The change in refractive index
alters the path of incident light enough so that the reflected
light has a different frequency [1]. Hence a shift in frequency
is correlated to presence of protein on the surface. The distant
resolution of CRR is similar to SPR and changes as small as 1
nm of surface height can be detected by both systems [1]. This
size range is small enough to be able to precisely detect
protein deposition or binding on the surface.
The special grating sensor is designed as depicted in the
following diagram:
Fig3. Schematic representation of the CRR sensor shows grating which is
produced using photolithography with dimensions of similar magnitude as
wavelength of visible light.
Both the incident and reflected light are perpendicular to the
surface. The grating size is a fraction of light wavelength and
as such only a single frequency is reflected back. The
advantage of CRR detection is that it can be adapted to highthroughput screening. For example a fiber optic can activate
and scan many spots in a fraction of second, effectively
producing real-time detection of protein binding [1].
Furthermore, since a special prism is not required for
detection, sensor fabrication is easier.
Detection and labeling problems are simultaneously solved
by approaches such as SPR and CRR where label free, realtime and precise detection is possible. Furthermore,
commercialized versions of these sensors are becoming
available. Biacore, British Windsor Scientific, Nippon Laser
and Electronic Laboratory, Texas Instruments, Coring and
SRU biosystems all have variation of these sensors, and are
developing platforms for high-throughput screening [3].
However, these approaches rely strongly on the presence of
highly specific tags so that only the protein of interest is bound
to the surface. As such in order to develop high-throughput
protein microarrays, a reliable tagging system is required.
IV. PROTEIN TAGS AND SOLVING THE PROBLEM OF DETECTION
Ideal tagging involves minimum cross binding and
maximum specific binding. For brevity, the most recent
antibody free system, which is easily adaptable to detection by
surface plasma resonance, is considered. Nucleic Acid
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Programmable Protein Array (NAPPA) involves introduction
of a protein-coding DNA segment on the surface of a
microarray and subsequent cell-free translation of the DNA to
the correct protein through use of enzymes such as T7
polymerase [5]. The translated protein includes a terminal tag
peptide, for which an antibody is present on the surface of the
array [5]. Hence the translated proteins are immobilized on the
surface of the array because the terminal peptide binds to the
antibody on the surface. The same antibody is used throughout
the array as it functions solely to hold the proteins on the
surface of the array. Using this method a library of different
proteins can be produced on the surface of the microarrays.
This method has several advantages. First, the same
photolithography techniques that are used to produce DNA
microarrays can in principle be used to make NAPPA arrays
[5]. Second, the NAPPA array can be stored without the
problem of protein denaturing because the un-translated array
only contains DNA and only through introduction of enzymes
does it become active [5]. Third, the production of NAPPA
array is financially feasible because most of the technologies
that make it possible are already commercially available.
V. MAKING THE PROTEIN MICROARRAY
Genes coding for antibodies in humans have a variable
region VL of less than 400 base pairs that codes for the
attachment site of the antibody to a protein, giving rise to its
high specificity [7]. It is possible, although difficult, to
produce this DNA segment on a NAPPA array. This can be
achieved by producing a linear stretch of DNA coding for the
VL region followed by the segment coding for the attachment
peptide using photolithography. The sequences for the VL
regions of most typical antibodies can be found in the Kabat or
NCBI database [8]. This linear sequence is then spliced using
enzymes and an exon is produced. Using cell-free translation
as is done in NAPPA arrays; protein of interest is produced on
the array. The protein is immobilized on the surface of the
array through attachment of its terminal peptide to the
antibody that is present on the array surface as in NAPPA. If
any step of this process does not take place successfully then
the translated protein will not bind to the surface antibody
because the terminal peptide is the last segment to be
translated. Hence it is possible to produce highly specific tags
in a high-throughput manner using a modified NAPPA array.
But the important difference between the NAPPA array and
the proposed arrays is that SPR is used instead of fluorescent
tags to detect the presence of proteins. There are already highthroughput versions of SPR being commercialized, in
particular by Biacore. SPR detection provides several
advantages over other techniques including CRR in this case.
First, detection can be real-time; this is useful for studies
involving dissociation rate constants and non-specific binding.
Second, SPR can be used to detect not only antibody
attachment, but also attachment of cytokines to receptors or
protein-protein interactions. Third SPR can be used to find rate
constants. For example, if the concentration of protein of
interest is very low, it will take a long time for equilibrium to
establish. A plot of protein on the surface vs. time will plateau
as equilibrium is reached, the longer the time, the lower the
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concentration of the protein. Furthermore, the initial rate of
change of this plot is directly proportional to the association
rate constant, and the rate of change when the protein in no
longer present in the solution is directly proportional to
dissociation rate constant. Knowing the concentration of the
protein from equilibrium, one can determine an effective
dissociation rate constant (Kd) for either protein-protein,
receptor-cytokine or protein-tag interaction. Furthermore, if
the surface tag is designed so that only phosphorylated
proteins attach, then rate of phosphorylation as a function of
time can be determined. As such, this modified NAPPA/SPR
protein microarray will provide quantitative information
regarding protein-protein interactions. This information is
crucial for the development of chemical kinetic based models.
Furthermore, interactions that are predicted using models such
as Bayesian networks can be verified using this protein
microarray.
VI. CONCLUSION
The proposed NAPPA/SPR protein microarray is a highthroughput, quantitative means to study protein-protein
interactions and is useful for the development and verification
of cell signalling models. With the rapid parallel progress that
is taking place in high-throughput technologies and cell
signaling models, the proposed protein microarray will be a
central tool for systems biology in the near future.
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