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High-density DNA microarray technology has played
a key role in the analysis of whole genomes and
their gene expression patterns. The use of oligonucleotide
or cDNA arrays to study many thousands
of individual genes is now widespread, with applications
ranging from the profiling of gene expression
patterns in whole organisms or tissues to the
comparison of healthy and pathological samples.
However, despite the success of DNA microarrays, it
is obvious that biological functions are executed by
biomolecules such as proteins rather than by DNA
itself. Therefore, protein biochips are emerging to follow
DNA microarrays as a possible screening tool to
identify any protein–ligand interactions. Traditional
methods for the analysis of proteomes include twodimensional
gel electrophoresis or chromatography
which, when combined with mass spectrometry,
enable large-scale separation and identification of
proteins, including many of their modifications.
These proteomic methods have been applied to
the comparative study of expression patterns. For
example, comparative studies have been carried out
of differentially developed states and of diseased
versus normal tissues [1,2] and even of related
pathogenic versus non-pathogenic organisms [3].
Such experiments can be referred to as ‘unbiased’ or
‘discovery-oriented’ proteomics and lead to large data
collections. They contrast with ‘system-oriented’
proteomics experiments, where only a defined subset
of proteins are analysed, such as a family of proteins
related by function or sequence or proteins
belonging to a common pathway. System-oriented
proteomics aims at observing changes in the concentration,
localisation or modification of particular
proteins of interest. Measuring such quantitative
changes requires the development of analytical
microarrays. Such analytical microarrays can consist
of immobilised antibodies that may capture defined
amounts of the corresponding antigen [4]. A second
task of system-oriented proteomics is the definition
of the biological role of a specific protein, which
may involve the identification of interacting proteins
or, in the case of enzymes, of specific sub-
strates.
Predominantly, protein biochips will play a role
in the discovery-oriented proteomics, but it is also
foreseen that they will be implicated in systemoriented
proteomics.
Angelika Lueking
Ruhr-University Bochum,
Medical Proteome Center,
Universitätsstraße 150,
D-44780 Bochum,
Germany
Dolores J.Cahill
Centre for Human
Proteomics,
Royal College of Surgeons in
Ireland,
Dublin 2,
Ireland
Stefan Müllner*
PROTAGEN,
Emil-Figge-Str.76a,
44227 Dortmund,
Germany
*e-mail:
stefan.muellner@protagen.de
Angelika Lueking,Dolores J.Cahill and Stefan Müllner
The human genome has been sequenced and the challenges of understanding the
function of the newly discovered genes have been addressed. High-throughput
technologies such as DNA microarrays have been developed for the profiling of gene
expression patterns in whole organisms or tissues. Protein arrays are emerging to
follow DNA chips as possible screening tools. Here, we review the generation and
application of microarray technology to obtain more information on the regulation
of proteins, their biochemical functions and their potential interaction partners.
Already, a large variety of assays based on antibody–antigen interactions exists. In
addition, the medical relevance of protein arrays will be discussed.
1359-6446/05/$ – see front matter ©2005 Elsevier Ltd. All rights reserved. PII:S1359-6446(05)03449-5
Protein biochips:a new and versatile
platform technology for molecular
medicine
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Chip content
For the generation of protein biochips, especially for the
discovery-oriented approach, large numbers of biomolecules
have to be produced. This is a major technical challenge,
calling for highly parallel, preferably automated
recombinant expression systems.
High-throughput sub-cloning of open reading frames
(ORFs) has been described for the genome of humans,
Saccaromyces cerevisiae, Arabidopsis thaliana and Caenorhabditis
elegans ([5–9]). Such recombination-based cloning approaches,
which lead to collections of individual cDNA
expression constructs, are strongly dependent on the
progress of genome sequencing projects and the annotation
of those sequences [10,11]. This means that uncharacterised
proteins will not be represented, limiting this
approach as a discovery tool. Additionally, clear determination
of the expressed sequence remains difficult
because of differential mRNA splicing. For these reasons,
this approach has proven most valuable for the production
of chips containing proteins from well-characterised
organisms, such as S. cerevisiae and C. elegans [6,8].
The effort of creating individual cDNA expression constructs
can be reduced by the generation of arrayed cDNA
expression libraries that generate thousands of cDNA
expression products in parallel [12–14]. By adding an
affinity tag (e.g. His tag or GST tag) to the 5′ end of the
cDNA insert, expression clones can be rapidly detected
and purified via the detection of their His- or GST-tagged
fusion protein. The automated recombinant expression
and purification is most frequently performed in E. coli,
which is a robust and convenient host organism [15–17].
However, many eukaryotic proteins end up in bacterial
cytoplasmic inclusion bodies and can only be recovered
in denatured form. When immobilising such denatured
proteins onto the microarray, the proteins display linear
sequence epitopes. Such a retained display of linear epitopes
is sufficient for antigen–antibody screening purposes
[18–21], but not for investigating the functionality or biological
role of proteins. Therefore, eukaryotic expression
systems, such as S. cerevisiae [7,8,22,23] or Pichia pastoris,
have been adapted to high-throughput expression and
purification [14,24,25]. In addition, these systems are able
to perform post-translational modifications of recombinant
proteins. Albala and co-workers have applied a baculovirus
expression system to the 96-well format and
expressed 72 different human cDNA clones in high-throughput
format, of which 42% produced a soluble product [26].
Similar to protein biochips, the generation of antibody
arrays requires a large number of high affinity, high specificity
protein binding ligands (e.g. antibodies), ideally one
for each protein of the proteome of interest. For the
human proteome, this means the generation of >100 000
protein binders if all the different post-translational modifications
are taken into account. Additionally, those
binders should be of high quality with respect to binding
behaviour, affinity and specificity. Polyclonal antibodies
might not be the best choice as their polyclonal state
means they might recognize different epitopes in proteins,
which could result in more cross-reactivity. Moreover,
obtaining thousands of poly- or monoclonal antibodies
by mouse immunisation is highly expensive and raises
ethical and patent-related questions. Therefore, the use
of recombinant antibodies is strongly recommended for
high density antibody arrays [27]. Antigen-binding fragments
such as Fab or ScFv provide simple antibody formats
that can be affinity selected in vitro by display technologies,
such as phage or ribosome display [28,29]. Alternatively,
aptameres, which are single-stranded oligonucleotides,
have been systematically evolved by an exponential
enrichment (SELEX) process to bind proteins [30]. They
appear to be promising new array probes as they can be
photo-crosslinked to the recognized proteins with very
low background from other proteins in the sample.
Because no other proteins are immobilised onto those
arrays, non-specific protein stains can be used to detect
the ligands [31,32].
Protein microarray applications
Protein arrays comprised of immobilised proteins are an
emerging biochip format. Protein biochips have been used
for protein–antibody and auto-antibody profiling, for the
study of protein–ligand interactions, where the ligand can
be either proteins, peptides, DNA or RNA, and for the
determination of enzymatic activity and substrate specificity
of classes of enzymes.
Protein–antibodyinteractionanalysis
The specificity and cross-reactivity of antibodies has been
successfully determined using high-content protein
biochips [20,33,34]. As antibodies are used extensively as
diagnostic and clinical tools, the characterisation of their
binding specificity is of prime importance. Additionally,
well-characterised antibodies are essential for the generation
of highly specific antibody arrays.
In functional immunomics, antibody–antigen interactions
are exploited in medically relevant contexts such as
autoimmune diseases. Screening protein arrays with sera
or plasma from auto-immune patients would not only
allow the identification of potentially new auto-antigens,
but also enable the diagnosis and sub-typing of the autoimmune
disease on the basis of the presence of specific
autoantibodies [20,35]. By combining the cDNA expression
library approach with protein microarrays, the humoral
auto-immune repertoire of dilated cardiomyopathy (DCM)
patients has been profiled and several protein antigens have
been determined which are associated with heart failure
[18]. In a different approach, a protein array consisting of
196 structurally diverse biomolecules representing major
autoantigens was probed with serum from patients with
different autoimmune diseases including systemic lupus
erythematosus (SLE), Sjögren syndrome and rheumatoid
arthritis (RA). There were distinct autoantibody patterns
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for the different autoimmune diseases, indicating their
suitability for diagnosis [35]. Using a mouse model of type
1 diabetes, a panel of 27 different antigens was determined
that allows discrimination between mice resistant or susceptible
to cyclophosphamide-accelerated diabetes [21].
It is suggested that autoantigen patterns as determined
by functional immunomics may allow predictive medicine.
For example, NOD mice spontaneously produce IgG antibodies
to the acetylcholine receptor, which is an antigen
of pathogenic antibodies in experimental autoimmune
myasthenia gravis (EAMG). At that time, there were no
reports that NOD mice were susceptible to EAMG induction.
To test if the presence of autoantibodies recognizing
the acetylcholine receptor has predictive potential, NOD
mice were challenged with a standard protocol used to
induce EAMG, and the mice developed EAMG [36]. It
has to be considered that in many cases patterns of
autoantigens/autoantibodies have not yet been assigned
to a function in the disease process, as functional studies
remain difficult. However, the specificity of the autoimmune
response may be an important tool for diagnosis,
classification and prognosis.
The diversity of the autoimmune response is a great
challenge for the development of antigen-specific tolerising
therapies. For example, an increased diversity of autoantibodies
in acute and chronic experimental autoimmune
encephalomyelitis (EAE), a mouse model for multiple sclerosis
(MS), is predictive of a more severe clinical course.
It has been shown that the use of DNA vaccines encoding
autoantigens such as epitopes of myelin prevent induction
of EAE [37–39]. To identify new autoantigens that
may act as tolerizing vaccines, an antigen microarray consisting
of 232 proteins specific for the myelin proteome
was generated and additional proteins were identified as
potential targets of autoimmune response in chronic EAE
[40]. Such analysis of immune response can also be
applied to other diseases. For example, to analyse the
humoral immune response to cancer, solubilized proteins
from the LoVo colon adenocarcinoma cell line were separated
into 1760 fractions, which were arrayed in microarray
format and incubated with sera from newly diagnosed
patients with colon cancer. One fraction, which exhibited
a strong reactivity to colon cancer sera, was subjected to
mass spectrometry, leading to the identification of a
putative antigen [41]. Another immediate application is
the use of ‘allergen arrays’ to screen for the presence of particular
IgE molecules in a patient sample. The traditional
approach involves the use of simple extracts from potential
allergens. These extracts are commonly used in skin
prick tests to determine the possible source of an allergic
reaction in the patient. By combining array technology
with recombinant allergens (e.g. pollen and fungus proteins),
large arrays have been produced that enable fast
screening of many allergens simultaneously [42–45].
These arrays can also readily include non-protein allergens
(e.g. latex).
Protein–peptideinteractionanalysis
Protein microarrays are suitable to study protein–ligand
interactions in which the ligand can be a protein, peptide,
DNA, RNA, oligosaccharide or chemical compound.
In a recent approach, the interaction of a restriction
enzyme to double-stranded DNA was monitored on
a micron-sized monolayer surface using atomic force
microscopy [46].
In a discovery-oriented approach, a yeast proteome
chip containing 5800 recombinant yeast proteins was
generated and used for the identification of known as well
as new calmodulin binders [8]. In addition, lipid binding
specificity was profiled using phosphoinoside-doped
liposomes [46].
In a system-oriented approach, the conserved cytoplasmic
motif KVGFFKR from the platelet membrane protein
integrin, which has previously been shown to play a critical
role in the regulation of activation of the platelet
integrin αIIb
β3
, was used for interaction studies [47,48].
The tagged peptide (biotin–KVGFFKR) was screened
against a high-density array of ~37 000 E. coli clones
expressing recombinant human proteins [12,33] and thirteen
different proteins were identified as binding the labelled
peptide (Figure 1). By peptide pulldown assays and coprecipitation
experiments, the interaction between a
putative chloride channel (ICln) and integrin αIIb
β3
has
been confirmed [47]. This experiment demonstrates the
enormous potential of a protein array approach, not only
for the identification of novel protein interactions, but
also for the teasing apart of biological pathways in general.
Protein–chemicalcompoundinteractionanalysis
For drug development, the analysis of the interactions
of chemical compounds or important pharmacological
targets with proteins is of major interest. For example,
the isoxazole derivative Leflunomide (N-(4-trifluoromethylphenyl)-5-methylisoxazol-4-carboxamide,
HWA
486) is a immunoregulatory and anti-inflammatory drug
with proven in vivo efficacy. Strong experimental evidence
points to the mitochondrial enzyme dihydroorotate dehydrogenase
(DHODH) as the major target of Leflunomide’s
mode of action [49]. Nevertheless, not all effects of the drug
can be explained by the interaction with DHODH. Therefore,
by using affinity chromatography, ten so far unknown
intracellular potential binding partners of the Leflunomide
were identified. Three of these proteins play a key role in
the second part of the glycolytic pathway. A further
validation by BIACORE® analysis revealed that Leflunomide
specifically bound pyruvate kinase, GAPDH, malic dehydrogenase
and lactic dehydragenase [50]. It is suggested
that protein arrays with high content may have a strong
impact on such interaction screening assays as the mode
of action of therapeutically interesting drugs may be more
deeply explained using these methods. However, additional
validation methods such as BIACORE® or pull-down assays
are essential.
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Enzymeactivityanalysis
Phosphorylation of proteins by protein kinases plays a
central role in regulating cellular processes and it is suggested
that this process contributes to many diseases,
including diabetes, inflammation and cancer. Therefore,
kinases are an important class of drug targets, resulting in
a strong interest in identifying new kinases and their substrates.
Different enzyme activities including phosphatases,
peroxidases, galactosidases, restriction enzymes and protein
kinases have been analysed on protein, peptide and
nanowell microarrays [7,51–59]. Zhu et al. have created
protein arrays of S. cerevisiae kinases [7]. In this study, a
total of 119 known or predicted protein kinases were
expressed, purified as GST fusion proteins, arrayed and
cross-linked on a protein chip and assayed with 17 different
substrates for auto-phosphorylation by treatment with radiolabelled
ATP, and new activities have been found [7]. For
example, 27 kinases show a phosphorylation activity of
poly-Glu-Tyr, indicating that many kinases are capable of
phosphorylating tyrosine even if they are members of the
serine/threonine family on the basis of sequence comparison.
Also, when comparing kinase activities across the
different substrates, many kinases were found to phosphorylate
one or two substrates. This result was confirmed
in another experiment, where the 768 purified proteins
acting as putative substrates for barley protein kinase
CK2α were immobilised onto the surface. Out of these
768 proteins, 21 potential substrates of CK2α were identified.
Most of these proteins represent high mobility group
proteins or calreticulin [59].
Conclusions
Applications of protein array technology, such as target
identification and characterisation, target validation,
diagnostic marker identification and validation, pre-clinical
study monitoring and patient typing seem to be feasible.
Proteins as drug targets dominate pharmaceutical R&D,
with ligand–receptor interactions comprising ~45% and
enzymes 28% of the targets [60]. Additionally, many therapeutic
proteins, especially humanized antibodies, are in
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FIGURE 1
Outline of the approach for identification of proteins that interact with the cytoplasmic tail of a membrane protein,in this case a platelet
integrin. (a) The structure of integrin αIIb
β3
is shown [65].(b) A tagged peptide (biotin-KVGFFKR) was synthesized.(c) This peptide was screened
against a high-density array of 37000 E.coli clones expressing recombinant human proteins.19 clones,coding for 13 different proteins,were
identified as binding the labelled peptide.(d) Peptide pulldown assays show a strong binding between this labelled peptide and purified proteins
isolated from three clones.Of these clones,one codes for a protein which could not be shown to be present in platelets,and two code for a putative
chloride channel,ICln.(e) Co-precipitation of ICln and platelet integrin has been successfully shown [64].
Drug Discovery Today
Cl channel probe
Integrin probe
Peptide pulldown assay
Integrin probe
Co-precipitation of chloride channel
and integrin αIIbβ3 from platelet lysate
Screening of 37000
human expression clones
(hEx1 library) reveals
binding of KVGFFKR
peptide to 19 clones –
including two coding for
a chloride channel.
-Biacore affinity studies
-In vivo co-localization
-Surface plasmon resonance
Peptide synthesis:
-KVGFFKR
-KAAAAAR control
Integrin IIbβ3
205
116
45
36
116
9
7
KAAAAAR control
KVGFFKR peptide
KVGFFKR
precipitation
KAAAAAR
precipitation
Beads + lysate
Lysate
+
M
ab
Cl Channel IP
Lysate
IP
Lysate
+
M
ab
Cl Channel IP
Lysis Buffer IP
Lysate
+
anti-α IIb
β 3
IP
(a) (c) (d)
(b)
(e)
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clinical development, such as Avastin®, Remicade® and
Enbrel®. The ultimate application for high-throughput
screening would be to test new leads or new targets in a
highly parallel manner. Some examples for applications
in this direction already exist. Recently, an immunosensor
array has been developed that enables the simultaneous
detection of clinical analytes [61]. Here, capture antibodies
and analytes were arrayed on microscope slides using
flow chambers in a cross-wise fashion. This current format
is low-density (6 × 6 pattern) but has high-throughput
potential, as it involves automated image analysis and
microfluidics; it is already being used as a format for
enzyme activity testing and other assays [62]. In another
study, small sets of active enzymes were immobilized in a
hydrophilic gel matrix. Enzymatic cleavage of the substrate
could be detected and inhibitors blocked the reaction [53].
More recently, an enzyme array that is suitable for assays
of enzyme inhibition has been reported [63]. Initial publications
in the area of receptor–ligand interaction studies
using a microarray format have shown that the interaction
of immobilized compounds and proteins in
solutions can be determined [8,50,56]. This technology allows
high-throughput screening of ligand–receptor
interactions with small sample volumes.
The multi-parallel possibilities of protein array applications
have the potential not only to allow the
optimization of pre-clinical, toxicological and clinical
studies through better selection and stratification of
individuals, but also to affect how diagnostics are used in
drug development.
REVIEWS
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