Front. Biol.

2012, 7(4): 336349

DOI 10.1007/s11515-012-1236-9

REVIEW

Functional protein microarray: an ideal platform for investigating

protein binding property

Shu-Min ZHOU1,2, Li CHENG1,2, Shu-Juan GUO1,2, Heng ZHU ( )3,4, Sheng-Ce TAO ()1,2
1
Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Jiao Tong University,
Shanghai 200240, China
2
State Key Laboratory of Oncogenes and Related Genes, Shanghai 200240, China
3
Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
4
The High-Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA

Higher Education Press and Springer-Verlag Berlin Heidelberg 2012

Abstract Functional protein microarray is an important tool for high-throughput and large-scale systems biology
studies. Besides the progresses that have been made for protein microarray fabrication, signicant advancements have
also been achieved for applying protein microarrays on determining a variety of protein biochemical activities. Among
these applications, detection of protein binding properties, such as protein-protein interactions (PPIs), protein-DNA
interactions (PDIs), protein-RNA interactions, and antigen-antibody interactions, are straightforward and have
substantial impacts on many research elds. In this review, we will focus on the recent progresses in protein-protein,
protein-DNA, protein-RNA, protein-small molecule, protein-lipid, protein-glycan, and antigen-antibody interactions.
We will also discuss the challenges and future directions of protein microarray technologies. We strongly believe that
protein microarrays will soon become an indispensible tool for both basic research and clinical applications.

Keywords lectin microarray, protein microarray, protein-cell interaction, protein-DNA interaction (PDI), protein-protein
interaction (PPI)

Introduction Delehanty and Ligler, 2003; Delehanty, 2004). Contact

printing is more popular for fabricating high-content protein
Protein microarrays, also known as protein chips, are microarrays. Quilled metal pins are usually used to deliver
miniaturized, parallel and high-throughput analysis systems subnanoliter of proteins to a slide surface by contact printing,
that are usually formed by spotting down hundreds to while noncontact dispensing techniques deliver a small
thousands of different proteins at high-density on a glass slide droplet of a protein sample to a slide surface without
(Zhu et al., 2001; Tao et al., 2007; Yang et al., 2011). Inherited touching it (Roda et al., 2000; Avseenko et al., 2002). For
the advantages of DNA microarrays, they allow the protein immobilization, an ideal microarray surface should be
simultaneous determination of biochemical properties with capable of reserving protein conformation, protein activities,
a variety of analytes only at the cost of a small amount of retaining more proteins, and possessing very low non-specic
samples in a single assay. background. There are a variety of functionalized substrate
Unlike DNA oligo microarrays that are usually formed via slides. On the basis of the immobilizing principle, they can be
in situ oligo synthesis, protein microarrays are typically classied into three types: covalent, afnity, and 3D surfaces
fabricated by spotting down proteins onto a substrate slide (MacBeath and Schreiber, 2000; Kusnezow et al., 2003). On a
using a standard contact (MacBeath and Schreiber, 2000; Zhu covalent surface the glass is usually derivatized with
et al., 2001) or a noncontact microarrayer (Jones et al., 1998; aldehyde, epoxy, or NHS moieties that can form a covalent
bound with primary amines of a protein. Obviously, proteins
are immobilized on the surface in a random orientation.
Received May 8, 2012; accepted June 4, 2012 However, when a glass slide is coated with afnity reagents,
Correspondence: Heng ZHUa, Sheng-Ce TAOb such as nickel-NTA, GSH-, or streptavidin, proteins with the
E-mail: [email protected], [email protected] corresponding afnity tag (e.g., Hisx6, GST, and biotin) are
Shu-Min ZHOU et al. 337

more likely to be oriented uniformly on the surface (Jeong et formed the rst proteome microarray, representing a major
al., 2011). To achieve a maximum amount of proteins breakthrough of the eld of protein microarray technology.
absorption on a surface, nitrocellulose- (Stillman and Recent years have witnessed a ourish of such proteome
Tonkinson, 2000; Kramer et al., 2004) and PAGE gel-coated microarrays in other organisms, such as viruses, bacteria, and
glass slides (Angenendt et al., 2002; Charles et al., 2004) have humans. In parallel, Snyder, Zhu, and other groups have
been developed and they do have unique advantages as we developed various types of biochemical assays, especially
will discuss later in this review. covalent enzymatic reactions, and demonstrated their appli-
Unlike nuclei acids, proteins have very different biochem- cations in both basic research and clinical applications
ical properties, thus, detection of protein binding properties (Nielsen et al., 2003; Ptacek et al., 2005). These enzymatic
has to be performed under the condition of minimum assays developed for protein microarrays have been exten-
destructive effect to protein conformation. Fluorescent dye sively reviewed elsewhere (Zhu and Snyder, 2001; Mac-
and radioisotopes are the two most frequently used labeling Beath, 2002; Chen and Zhu, 2006; Zhou et al., 2011). Herein,
strategies for signal detection (Shingyoji et al., 2005; Zheng et we focus on the latest applications of protein microarrays for
al., 2005; Koshi et al., 2006; Zajac et al., 2007). However, in the probing of protein binding properties (Kramer et al., 2004;
most of the cases, labeling is a tedious process, because some Hamelinck et al., 2005; Popescu et al., 2007a,b; Wingren and
of the analytes are very difcult or may even unable to be Borrebaeck, 2008) (Fig. 1).
labeled. Thus, the ideal strategy is labeling-free detection. To
achieve this, several approaches have been developed
recently, including scanning ellipsometry (Carlsson et al., Protein-protein interactions
2005), surface plasma resonance (SPR) (Mecklenburg et al.,
2002), resonance light scattering (Gao et al., 2010), and the Protein-protein interactions (PPIs) are of central importance
oblique-incidence reectivity difference (OI-RD) method for virtually every biologic process. There are numerous
(Zhu et al., 2007a). technologies for discovering and measuring PPIs. Yeast two-
In 2001, the Snyder group (Zhu et al., 2001) reported a hybrid (Y2H) is a classic technology for screening novel
protein microarray composed of 5800 individually puried, protein-protein interactions. Alternatively, phage display-
unique yeast proteins. Because this microarray covers more based screening has also been widely applied for this
than 80% of the proteins encoded by the yeast genome, it purpose. The Y2H approach has a tremendous success in

Figure 1 Protein microarrays for protein-ligand interaction study. The ligands could be probed on the protein microarray are protein,
DNA/RNA, small molecule, glycan, lipid and etc.
338 Functional protein microarray: an ideal platform for investigating protein binding property

construction of PPI networks in a variety of model organisms;

however, they are quite laborious to perform proteome-wide
because a minimum of N2/2 combinations of Y2H assays
have to be carried out for a given organism encoding a
number of N proteins. Furthermore, the associated high false
positive rate normally requires reciprocal conrmation
assays, which would further increase the labor of Y2H assays
substantially. Finally, these approaches only provide binary
results they cannot provide any information about a given
PPI afnity value.
On the other hand, the protein microarray technology may
serve as a complementary method for PPI screens as it is
capable of screening protein-protein and protein-peptide
interactions in high-throughput fashion with the capability
of quantication.
In general there are three slightly different strategies for
studying protein-protein interactions on a protein microarray
(Fig. 2). When a highly specic antibody with high afnity
against the native protein in question is available, positive PPI
events can be detected using a uorescent conjugated
antibody (Fig. 2a). When the protein in question is fused
with an afnity tag, such as GST, 6xHis, and V5, antibody
against that tag can be used, followed by incubation with a
uorescently labeled second antibody (Fig. 2b). In the last
scenario, if a protein in question does not have a specic
antibody available and without any tag, the protein can be
labeled directly with a uorescent dye or biotin. However, the
labeling efciency is critical for the nal binding results; an
optimal labeling assay is normally required (Fig. 2c).
For example, potential binding partners of calmodulin were
surveyed against the yeast proteome microarray as reported Figure 2 Labeling strategies for protein microarray based PPI
by Zhu et al. (2001). By incubating biotinylated calmodulin study. (a) Bound target proteins are recognized by a specic
with calcium on the microarrays, six of the 12 known primary antibody followed by a uorescent secondary antibody or
by a biotinylated secondary antibody. (b) The target protein is
calmodulin binders were successfully identied; of the other
prepared as a fusion protein with an afnity tag, thus the binding
6, two are not in their collection thus not on the protein could be readout by a primary antibody specic for the afnity tag
microarray, and the other 4 are not detectable in the quality followed by a uorescent secondary antibody. (c) The target
control experiment (GST probing). They also discovered 33 protein could be directly labeled by a uorescent dye when there is
novel potential calmodulin binding proteins, and sequence no specic antibody and afnity tag available.
analysis revealed that 14 of the 39 colmodulin binder have a
consensus sequence, presumable a novel calmodulin binding biophysical interactions were found. In addition, they
motif. discovered different receptor tyrosine kinases become pro-
The real power of protein microarrays in PPI studies was miscuous differently when overexpressed. To our knowledge,
demonstrated by MacBeath and colleagues using a protein this systematic study demonstrated that protein microarrays
microarray composed of puried protein domains (Jones et could also be used for quantitative (at least semiquantitative)
al., 2006). They developed a binding assay that allowed for measurement of protein-ligand interactions.
semiquantitative measurement, and applied it for investigat- To evaluate the performance of protein microarrays in PPI
ing protein-peptide interactions which play an important role study, it is required to compare it side-by-side with other
in signaling pathways. Sixty one peptides representing platforms. Tao group selected an apoptosis related protein
tyrosine phosphorylation sites on four ErbB receptors were BAG3 as an example and screened for its binding partners
incubated with the array containing 159 human Src homology using both a human proteome microarray with > 17000
2 (SH2) and phosphotyrosine binding (PTB) domains. Eight human proteins (Jeong et al., 2012) and a mass spectrometry-
concentrations of each peptide, ranging from 10 nM to 5 mM, based strategy simultaneously. Surprisingly, most of the
were tested in the assay, allowing quantitative measurement known binders have been successfully identied by both of
of the binding afnity of each peptide to its protein ligand. As the two platforms, and more impressively, more than 50% of
a result, 43 previously recognized interactions and 116 new the newly discovered BAG3-interacting proteins were
Shu-Min ZHOU et al. 339

identied as shared targets using both two platforms. These mobility assays, are frequently applied for this purpose.
results indicate that protein microarray is comparable with Recently, chromatin immunoprecipitation (ChIP) coupled
mass spectrometry for PPI study while much less time and with either DNA microarrays (ChIP-chip) or deep sequencing
labor is required (Manuscript in preparation). (ChIP-seq) as detection methods have ourished in globally
In another application, Popescu et al. developed a protein identifying genomic sites recognized by a particular TF in
microarray containing 1133 proteins in Arabidopsis thaliana vivo. Because of its simplicity, capability of parallel and high-
and also applied this microarray for discovering proteins that throughput analysis, protein microarrays are also suitable for
could bind to calmodulins or calmodulin-like proteins global proling of protein-DNA/ RNA interactions. Specic
(Popescu et al., 2007a). To identify CaM/CML-interacting applications include DNA repair protein discovery, DNA
proteins, the protein microarrays were probed with Alexa motif-protein interaction network construction, and viral
Fluor 647-conjugated CaM1, CaM6, CaM7, CML8, CML9, RNA-host protein interactions (Fig. 3) (Hall et al., 2004;
CML10, CML12 in the presence of calcium, and an Alexa Chen et al., 2008; Hu et al., 2009).
Flour 594-conjugated bovine CaM (BtCaM) was used as a In the earliest protein-DNA interaction study on a protein
positive control. A total of 173 different proteins that bound microarray, Snyder and colleagues screened for novel DNA
the three CaMs and four CMLs were identied. Approxi- binding proteins by probing the yeast proteome microarrays
mately 25% (44 of 173) of the proteins interacted with all with uorescently labeled, fragmented yeast genomic DNAs
CaMs/CMLs, whereas the same percentage of proteins (Hall et al., 2004). Among the ~200 potential DNA binding
interacted with only one CaM/CML. The remaining 50% of proteins identied in this study, half of them were previously
the proteins bound to two or more CaMs/CMLs. Interestingly, not known to be able to bind to DNA molecules. Further
a large number (60 of 173) of CaM/CML-interacting proteins functional studies (e.g., ChIP-chip, knockout, and EMSA)
were transcription factors. Bioinformatics analysis showed showed that one of such proteins, Arg5,6 that encodes two
that CaM binds to several TGA and WRKY transcription catalytic enzymes in ornithine biosynthesis, could also bind to
factors that play a role in the activation of stress or defense a specic DNA motif and plays a role in the regulation of both
pathways. Furthermore, 20 kinase or kinase-like proteins transcriptional and posttranscriptional processes. The dis-
were also identied, implying that they might be substrates of covery of such a moonlighting function of Arg5,6 as a TF was
various CaMs/CMLs. only possible with the unbiased global screening, which is
The latest example of applying protein microarray for one of the key feature of protein microarray technology. Later,
systematic PPI study was reported by the Snyder group. To the Snyder and Johnston groups have fabricated a protein
fully and systematically understand the pathways that protein microarray with 282 known and predicted yeast TFs and
kinases are involved, they puried 85 out of the 122 yeast probed the microarray with 75 DNA motifs individually (Ho
protein kinases and probe them individually on a yeast et al., 2006). Over 200 specic PDIs were identied
proteome microarray (Fasolo et al., 2011). One thousand and and > 60% of them are previously unknown. Most impor-
twenty three PPIs were successfully identied and most of tantly, the DNA binding activity of Yjl103p, a previously un-
these PPIs are novel. Further bioinformatics analysis revealed characterized DNA binding protein was conrmed, and many
that many of these PPIs could be linked to previously distinct of its target genes were also identied. Further analysis
cellular pathways. Overall, their results indicate that kinases showed that most of these genes are involved in stress
operate in a highly interconnected network that coordinates response and oxidative phosphorylation.
many activities of the proteome. To identify new DNA damage/repair related proteins, Chen
et al. (2008) fabricated a bacterial proteome microarray
containing 4256 proteins encoded by the E. coli K12 strain,
Protein-DNA and protein-RNA interactions which covering: 99% proteins of the proteome. End-labeled,
double-stranded (ds) DNA probes carrying abasic or
Protein-DNA interactions (PDIs) play a central role in mismatched base pairs were probed on the microarrays. A
regulating DNA replication and gene transcription. In small number of proteins were specically recognized by
general, proteins that directly bind to DNA in a sequence- each type of the probes with high afnity. Two of them
specic fashion and are capable of regulating nearby gene (YbaZ, YbcN) were further characterized to have base-
expression (either activation or repression) are considered as ipping activity.
transcriptional factors (TFs) (Teichmann and Babu, 2004; Because of the importance to fully understand transcription
Popescu et al., 2007b). In addition, sequence-specic DNA regulation, several new approaches have been recently
binding proteins other than TFs may also be very important in developed to proling PDI specicity. Bulyk and colleagues
DNA replication, DNA repair, and chromosome dynamics developed a ds-DNA microarray that covers the entire DNA
(Zhu et al., 2003; Petukhova et al., 2005). 10-mer space and applied it to determine consensus motifs of
The most intensively studied subset of PDIs is those TFs (Berger and Bulyk, 2009). Wolfe and colleagues applied
between DNA binding proteins and their specic DNA target the yeast/bacteria one-hybrid approach for the same purpose
sequences. In vitro assays, such as DNA footprinting and gel (Meng and Wolfe, 2006). Although highly successful, such
340 Functional protein microarray: an ideal platform for investigating protein binding property

Figure 3 Protein microarrays for protein-DNA/RNA interaction study. Generally, DNA or RNA molecules are end-labeled and proled
on a protein microarray. After washing away the non-specic binding DNA/RNA, the interactions between protein and DNA/RNA can be
detected and recorded by a microarray scanner. A variety types of probes such as damaged DNA, DNA motif and virus shRNA could be
used as analyte.

DNA-centered approaches require knowledge as which TFs kinases, and mitochondrial proteins. They probed the
to be studied. Because of this limitation, it becomes quite microarray with 400 predicted and 60 known DNA motifs.
challenging when the task is to identify potential proteins that 17718 PDIs were thus identied. Many known PDIs for TFs
specically recognized a DNA motif of interest, such as were successfully recovered. A large number of new PDIs for
motifs predicted to be functional as part of the effort in the annotated and predicted TFs were also identied. Over 300
ENCODE project (The ENCODE (ENCyclopedia Of DNA proteins that were not previously annotated as TFs were
Elements) Project, 2004). To solve this problem, Hu et al. found to show sequence-specic PDIs, including RNA
(2009) fabricated a protein microarray composed of 4191 binding proteins, mitochondrial proteins, and protein kinases
full-length human proteins, most of which are TFs, DNA- and (Hu et al., 2009; Xie et al., 2010). The most surprising nding
RNA binding proteins, chromatin-associated proteins, protein is from Erk2, a well studied MAP kinase. Using a series of
Shu-Min ZHOU et al. 341

well-designed in vitro and in vivo approaches, they demon- the protein microarray based small molecule-protein interac-
strated that this kinase had a capability of binding DNA tion studies. Before probing on the microarray, the compound
independent of its protein kinase activity. Further results has to be chemically conjugated with a biotin, a uorescent
revealed it act as a transcription repressor of transcripts dye or a radioisotope. However, the radioisotope labeled form
induced by interferon gamma signaling (Hu et al., 2009). is not available for most of the compounds. Even biotinyla-
Again, this study proved the unprecedented unbiased global tion and uorescent conjugation are difcult for the majority
screening capability of protein microarray for novel functions of the compounds, because there is either no appropriate site
of the un-characterized proteins as well as the moonlighting for labeling or the stability and original activity is ruined after
function of the well-studied proteins. labeling, or the labeling cost is unacceptable. The ideal
In addition to protein-DNA interactions, protein-RNA solution to solve this difculty is to combine protein
interactions are also very important for the regulation or microarray with a label-free technology. Bore this in mind,
inhibition of a variety of biologic functions. To test the Kenyon et al. coupled ligand-protein binding assays on a
feasibility of protein microarrays for protein-RNA interaction protein microarray and the MS/MS technology as the
study and screen for RNA binding proteins with antiviral detection method to detect antibody-epitope interactions.
activities, a uorescently tagged small RNA hairpin contain- Although this approach is still in its infancy, we expect that it
ing a clamped adenine motif, which is required for the will have a tremendous impact on drug target discovery when
replication of brome mosaic virus (BMV) was incubated with MS/MS sensitivity is further improved (Fig. 4 right) (Evans-
yeast protein microarray (Zhu et al., 2007b). The rationale for Nguyen et al., 2008).
using yeast proteome microarray is that this virus can also
replicate in Saccharomyces cerevisiae. Two of the candidates,
pseudouridine synthase 4 (Pus4) and the actin patch protein 1 Lectin-glycan interactions
(App1), were further validated in Nicotiana benthamiana, the
results showed that they can dramatically inhibit the spread of As one of the most diverse posttranslational modication
BMV in plants. (PTMs) of proteins, glycosylation is estimated to modify over
50% of proteins expressed in eukaryotes (Apweiler et al.,
1999). In particular, most of the plasma membrane-localized
Protein-small molecule interactions and secreted proteins, which encompass about one third of
eukaryotic proteomes, are heavily glycosylated. Thus, cell
Here we refer small molecules as drug or drug candidates. surface is coated with thousands of glycans, which mediate a
Usually, it costs more than one billion US dollars and take variety of biologic processes, such as cell-to-cell commu-
1215 years to bring a new drug to the market. Lots of nication, cell-matrix interactions, host-pathogen interactions,
potential compounds even failed at the nal step, i.e., phase development, tumor invasion and metastasis. Alters in the
III clinical trial. Ideally, if we can correctly lter out the composition or structure of these glycans may reect the
hopeless candidate compounds as many as possible at the dramatic changes in cell phenotype and signaling transduc-
early stage, much of the cost and time could be saved, thus tions inside cells. These changes could even cause diseases,
making the drug development process more fast and effective. such as the I-Cell disease (Kollmann et al., 2010) and
One possible solution for this is to prole the binding proteins leukocyte-adhesion deciency type II (LAD II) (Gazit et al.,
of a given compound and try to understand its molecular 2010).
mechanism on a systems level. This may also be useful for There are a handful of tools and technologies that can be
old-drugs as to explore the possibility for new treatment/s applied for glycosylation study, e.g., liquid chromatography
and to avoid its side-effects. (LC) (Hase et al., 1978; Tomiya et al., 1988), mass
Among many available technologies, protein microarrays spectrometry (MS) (Kameyama et al., 2005), capillary
may be the best choice for identifying the compound- electrophoresis (CE) (Kamoda et al., 2006; Kamoda and
targeting proteins globally because of its simplicity (Fig. 4). Kakehi, 2006), and ow cytometry (FCM) (Frojmovic et al.,
Huang et al. (2004) incubated biotinylated small-molecule 1991; Poulain et al., 1999). However, none of these
inhibitors of rapamycin (SMIRs) on the yeast proteome traditional approaches could meet the criteria of an ideal
microarrays, and identied several target proteins of the glycosylation study platform, which is economical, miniatur-
SMIRs, including Tep1p and Ybr077cp (Nir1p). Both of them ized, sensitive, and high-throughput. Borrowing the idea of
were validated in vivo and found to be associate with PI(3,4) protein microarrays, several groups have developed a
P2, which is a second messenger involved in membrane powerful tool for proling cell surface glycans by spotting
translocation of a variety of cell growth and survival signals. down lectins, a group of proteins that specially recognize
This suggests a novel mechanism by which phosphatidyli- various types of glycans, in microarray formats (Angeloni et
nositides might modulate the target of rapamycin pathway. al., 2005; Pilobello et al., 2005; Zheng et al., 2005) (Fig. 5).
The labeling of the compound is critical for the success of Kuno et al. (2009) developed an antibody-assisted lectin
342 Functional protein microarray: an ideal platform for investigating protein binding property

Figure 4 Protein microarrays for protein-small molecule interaction study. Usually, a small molecule such as drug candidate is
biotinylated or uorescent conjugated and probed on a protein microarray. As to overcome the shortcoming of chemically labeling of
small molecule prior to probing, MALDI-TOF may be applied as a readout technology. Briey, a protein microarray is fabricated on a
hydrophilic/hydrophobic patterned substrate slide. A mixture of un-labeled small molecules could be probed on this microarray, the results
were then readout by MALDI-TOF analysis.

proling (ALP) to detect glycoproteins at very low microarray, Hirabayashis group also developed evanescent-
concentration in clinic samples using lectin microarrays. eld activated uorescence detection system (Kuno et al.,
Using ALP, proteins were enriched by aid of the appropriate 2005; Uchiyama et al., 2006), on which the interactions
antibodies, which made it possible to be analyzed by a lectin between the analyte and lectins are monitored on the glass
array. They applied this method to analyze the glycan surface immersed under buffer that enables real-time detec-
structures of protein hPod, which has been proposed to tion of uorescently labeled glycans. Since the evanescent
enhance the metastatic potential of glioblastoma cells. Trying eld is generated within 200 nm, the background level is
to establish a wash-free detection platform on lectin extremely low and washing steps can be omitted.
Shu-Min ZHOU et al. 343

Figure 5 Live cell surface glycan proling using lectin microarray. Live cells from cell cultures or clinical samples are labeled with a
uorescent dye, such as CFSE. The labeled cells were probed directly on a lectin microarray. After incubation for a while, a mild washing
step should be taken to remove the non-specic bindings. The positive binding could be readout and recorded by a high-resolution
uorescence scanner.

To analyze the interactions between protein and glycopro- to analyze closely related E. coli strains, the nonpathogenic
teins in a higher level, intact cells including bacteria, fungi strains JM101 and HB101 and the pathogenic strains RS218.
(Kuno et al., 2005; Hsu and Mahal, 2006; Hsu et al., 2006), The results revealed that pathogenic bacterial strains had
and mammalian cells were proled on lectin microarrays, by distinct binding signals and different binding patterns from
which the dynamics of cell surface glycosylation during those of non-pathogenic trains, which could provide a surface
growth, and after stimulation were monitored (Pilobello et al., glycan ngerprint of bacteria (Hsu and Mahal, 2006; Hsu et
2005; Ebe et al., 2006; Chen et al., 2007; Pilobello and Mahal, al., 2006).
2007; Tateno et al., 2007) (Fig. 5). However, more exciting results came from the studies of
In the rst case of detecting living cells using lectin the mammalian cell surface glycans. In 2007, Ebe and Tateno
microarray, Hsu et al. fabricated a microarray with 21 lectins found signicant differences in carbohydrate expression on
344 Functional protein microarray: an ideal platform for investigating protein binding property

normal Chinese hamster ovary (CHO) cells and lymphatic probe the yeast proteome microarray. As a result, they
endothelial cells (LEC) with their different glycosylation identied more than 150 proteins binders of these phospho-
defective mutants using a microarray with 43 lectins (Ebe et lipids, more than 50% of which were previously unknown as
al., 2006; Tateno et al., 2007). In another interesting study, membrane-associated proteins. In a recent follow up study,
normal and tumorigenic human breast cell lines including the Lemmon group further analyzed those unconventional
their sublines differing in the tendency to home to different phospholipid binding proteins identied in the above study,
tissues during metastasis were analyzed by lectin microarray and found a new KA domain among three kinases, Kcc4,
and signicant differences in carbohydrate expression were Hsl1, and Gin4. Mutation in the KA domain impairs
observed (Chen et al., 2007). membrane association of the intact proteins and reveals the
In 2008, a lectin microarray with 94 different lectins was importance of phosphatidylserine for bud neck localization of
developed by Tao et al. To qualitatively prole the lectin yeast Kcc4p (Moravcevic et al., 2010). These results
binding specicity of 24 human cell lines, they developed a established protein microarray as a reliable tool for global
binary algorithm that generates a glycocode for each cell protein-lipid interaction study, as to improve the applicability
lines. This has allowed them to hierarchically cluster these of this tool, protocol for making liposomes with higher
cell lines on the basis of their accessible glycan composition. stability and more intensive labeling (uorescence dye or
Importantly, they also successfully identied three new lectin biotin) is highly anticipated. More recently, Chen and
biomarkers (LEL, AAL and WGA) that differentiate breast colleagues developed an non-quenchable liposome as a
cancer stem-like cells from their parental cell line MCF7. To carrier to identify yeast proteins that can specically bind to
further verify the results, an murine model was tested, the PI(3,5)P2 using the yeast proteome microarrays (manuscript
results showed that lectin-enriched cancer stem cells from submitted).
MCF7 cell population could indeed produce bigger and more
aggressive tumors (Tao et al., 2008). This study is the rst
case to show the potential of lectin microarray for live cell Antigen-antibody interactions in biomarker
specic marker identication. discovery
In a most recent study, Tateno et al. (2011) fabricated a
lectin microarray with 96 lectins and performed a compre- Because the majority of proteins present in a puried form on
hensive analysis of 114 types of human induced pluripotent a proteome microarray, they are ideally suited for examining
stem cells (iPSCs), which were generated from 5 types of antibody specicity, which is the biggest challenge for
somatic cells. Nine human embryonic stem cells (ESCs) were traditional antibody generating. In principle, a highly specic
also included in this lectin microarray probing. Data analysis antibody should recognize only its target among the other
showed that using the glycan binding proles, the undiffer- thousands of proteins on the microarray, and when it does not,
entiated iPSCs and ESCs could be clustered as one large its cross-reactivity to non-specic targets can be revealed.
group, which was clearly different from that of the This idea was rst tested by Michaud et al. who probed the
differentiated SCs. Further analysis showed that the expres- yeast proteome microarrays with 14 different commercial
sion proles of relevant glycosyltransferase genes agreed well antibodies: 6 monocolonal antibodies (mAbs), 6 anti-peptide
with the lectin microarray results. These results indicated that polyclonal antibodies, and two polyclonal antibodies raised
lectin microarrays could also be used as a reliable tool to against full length proteins (Michaud et al., 2003). The results
differentiate highly related cells during differentiation based were quite astonishing: the mAbs exhibited higher specicity
on their glycan proles. than their polyclonal counterparts, while the anti-peptide
polyclonal antibodies were more specic than those against
full-length proteins. However, even some mAbs showed
Protein-lipid interactions some off-target reactivity. This study raised the importance of
using proteome microarrays to identify mono-specic anti-
Protein-lipid interaction is an essential feature of biologic bodies. The use of protein microarrays to prole antibody
membranes. The protein-lipid interactions could also be binding proles has prompted two other interesting applica-
assayed on a protein microarray. However, due to the tions in mAb generation (Zhu et al., 2007a; Jeong et al.,
hydrophobicity of lipids, they could not be probed directly. 2012). In the past, complex biologic samples, such as cancer
They should be assembled into a liposome, which acts as a tissues, have been used as antigens to immunize mice to
carrier. Usually, each liposome contains phosphatidylcholine develop antibodies specic for the diseases. However, this
(PC), target lipids and biotin or uorescent DHPE for results approach only had limited success because it demands
readout. Alternatively, the uorescent dye can also be packed additional steps to identify the corresponding antigens, either
inside the liposomes (Fig. 6). In 2001, Zhu et al. (2001)used by screening a cDNA expression library or immunoprecipita-
ve types of PI liposomes which containing 5% (w/w) PI(3)P, tion (IP) coupled with MS. In a report by Hu et al. (2007) a
PI(4)P, PI(3,4)P2, PI(4,5)P2, or PI(3,4,5)P3 respectively to human protein microarray composed of 1000 individually
Shu-Min ZHOU et al. 345

Figure 6 Protein microarrays for protein-lipid interaction study. A target lipid is assembled into liposome with a biotinylated DHPC or a
uorescent dye. Except the sample preparation, the protein microarray based procedure for protein-lipid interaction study is similar to that of
protein-small molecule study.

puried proteins were used to determine antigens of Perspectives

hybridomas generated via immunizing mice with live cancer
tissues. The protein microarrays serve not only as a tool to Protein microarray technology has been shown to be very
deconvolute antigen-antibody interactions, but also as a way useful for multiplexed detection and proteomics studies,
to estimate specicity of the obtained mAbs, though among a especially for protein-ligand interaction studies. Novel
limited number of 1000 proteins. This idea was further applications utilizing protein microarrays and new protein
streamlined in a report by Jeong et al., where the human microarray technologies are continually emerging. However,
proteome microarrays composed of ~17000 full-length there are still several disadvantages that need to be overcame
proteins were used to deconvolute mAbs generated with before protein microarray technology could be widely applied
live cell immunization (Jeong et al., 2012). By performing a for protein-ligand interaction studies.
rather thorough characterization of these mAbs, they found First, the availability of protein microarray is still an issue.
that a large fraction of mMAbs are both of immunoblot- and The traditional cloning-expressing-purication-printing
immunoprecipitation-grade. approach is still the gold standard for making protein
346 Functional protein microarray: an ideal platform for investigating protein binding property

microarrays, especially for proteome microarrays. Because of Research of China (No. 2010CB529205), the National Natural
the sophisticated expertise required and the high cost of Science Foundation of China (No. 31000388), and funding support
production, it is almost impossible for most laboratories to to HZ from the National Institute of Health (CA160036, RR020839,
make their own microarrays, but the prices for the commercial DK082840, CA125807, and GM076102).
protein microarrays are unacceptably high. A variety of
promising strategies have already been tested to bypass the Abbreviations
traditional procedure (Ramachandran et al., 2004; Tao and
Zhu, 2006; He et al., 2008). However, none of them are close PPI: protein-protein interaction; PDI: protein-DNA interaction;
to be applied for large-scale fabrication of protein micro- GSH: Glutathione; NHS: N-Hydroxysuccinimide; SH2: Src homol-
arrays. Thus, to make protein microarray technology more ogy 2; PTB: phosphotyrosine binding; TF: transcriptional factor ;
applicable, a simpler and more powerful strategy is needed. ChIP: chromatin immunoprecipitation; BMV: Brome Mosaic Virus;
Before an alternative strategy can replace the current one, we SMIR: small-molecule inhibitors of rapamycin; MALDI-TOF:
envision that the current strategy will still be the mainstream matrix assisted laser desorption ionization/time of ight; PTM:
in the near future. The foundation of the traditional strategy is posttranslational modication; ALP: antibody-assisted lectin prol-
the high-quality and expression-ready ORF collections. ing; iPSC: induced pluripotent stem cell; ESC: embryonic stem cell;
However, there are only a handful of such collections, such PC: phosphatidylcholine; DHPE: N-1,2-dihexadecanoyl-sn-glycero-
as E. coli (Ogura et al., 2006; Chen et al., 2008), yeast (Zhu et 3-phosphoethanolamine; HUPO: Human proteome organization;
al., 2001; Gelperin et al., 2005; Fasolo et al., 2011), human
(Invitrogen, ProtoArray), Arabidopsis thaliana (Popescu et
al., 2007a, 2009). To accelerate biologic research and clinical
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