Protein microarrays as a discovery tool for studying protein–protein interactions

Exploring the function of the genome and the encoded proteins has emerged as a new and exciting challenge in the postgenomic era. Novel technologies come into view that promise to be valuable for the investigation not only of single proteins, but of entire protein networks. Protein microarrays are the innovative assay platform for highly parallel in vitro studies of protein–protein interactions. Due to their flexibility and multiplexing capacity, protein microarrays benefit basic research, diagnosis and biomedicine. This review provides an overview on the basic principles of protein microarrays and their potential to multiplex protein–protein interaction studies.     

Microarrays to characterize protein interactions on a whole-proteome scale

Protein microarrays contain a defined set of proteins spotted and analyzed at high density, and can be generally classified into two categories; protein profiling arrays and functional protein arrays. Functional protein arrays can be made up of any type of protein, and therefore have a diverse set of useful applications. Advantages of these arrays include low reagent consumption, rapid interpretation of results, and the ability to easily control experimental conditions. The ultimate form of a functional protein array consists of all of the proteins encoded by the genome of an organism; such an array would be the whole proteome equivalent of the whole genome DNA arrays that are now available. While proteome microarrays may not have reached the stage of maturity of DNA microarrays, recent developments have shown that many of the barriers holding back the technology can be overcome. Arrays of this type have already been used to rapidly screen large numbers of proteins simultaneously for biochemical activities, protein-protein interactions, protein-lipid interactions, protein-nucleic acid interactions, and protein-small molecule interactions. Eventually, functional protein arrays will be used to facilitate various steps in the drug discovery and early development processes that are currently bottlenecks in the drug development continuum.     

Applications of DNA and protein microarrays in comparative physiology

DNA microarrays have revolutionized gene expression studies and made large-scale parallel measurement of whole genome expression a feasible technique in model species where genomes are well characterized. Such studies are perfectly suited to unraveling the complex regulation and/or interaction of both genes and proteins likely involved in most physiological processes. Gene expression profiles are currently being used to identify genes underlying a range of physiological responses. Characterization of these genes will help to elucidate the pathways and processes regulating physiological processes. Expanding the use of DNA microarrays to non-model species that have been critical in elucidating certain physiological pathways will be valuable in determining the genes associated with these processes. Approaches that do not require complete genome information have recently been applied to "non-model" organisms. As whole genomes are sequenced for non-model organisms, the application of DNA microarrays to comparative physiology will expand even further. The recent development of protein microarrays will be critical in understanding the regulation of physiological processes not accounted for at the genomic level. Together, DNA and protein microarrays provide the most thorough and efficient method of understanding the molecular basis of physiological processes to date. In turn, classical physiological approaches will be vital in characterizing and verifying the function of the novel genes identified by microarray experiments. Ultimately, DNA and protein microarray expression profiles may be used to predict physiological responses.     

一种新型细胞微阵列的制作和应用

为了高通量地检测大量培养细胞中基因原位表达, 发明了一种制作细胞微阵列的新方法,成功地制作含20种细胞系共100个供体细胞石蜡混合物点阵的细胞微阵列.免疫组化检测P53, P21, PTEN、P16基因在细胞微阵列中的蛋白质表达.原位杂交检测BRD7、NGX6 基因在细胞微阵列中mRNA原位表达.建立了P53、P21、PTEN、P16蛋白和BRD7、NGX6 mRNA 在不同培养细胞中的原位表达谱.细胞微阵列为基因功能研究提供一种新的高通量工具.细胞微阵列可广泛用于DNA、RNA和蛋白质水平上的基因原位表达研究.细胞微阵列还可用于筛选药物作用靶标的研究.     

Using protein microarrays to study phosphorylation-mediated signal transduction

Unraveling the complexity of cell regulatory systems and monitoring their operations under normal and pathological circumstances is one of the major outstanding biomedical challenges. The phosphoproteome has emerged as a rich source of biomarkers for tracking cell signaling and disease, and many of the kinases that phosphorylate proteins represent attractive targets for drug development. Over 100,000 phosphorylation sites distributed in most of the 23,000 proteins encoded by the human genome have already been identified in a non-targeted fashion by mass-spectrometry. Antibody microarrays permit ultra-sensitive, semi-quantitative measurements of the levels of hundreds of target proteins and their phosphorylation in parallel with specimens from cells and tissues. Conversely, reverse-phase protein microarrays (RPPMs) that are printed with crude cell/tissue lysates allow tracking of a target protein with a probing antibody in hundreds to thousands of cell and tissue samples simultaneously. While more than half a million commercial antibodies are available, the identification of highly specific and potent antibodies for use in microarrays remains a major impediment. Antibody cross-reactivity is an issue for both antibody microarrays and RPPMs. The low abundance of signal transduction proteins and their substoichiometric levels of phosphorylation are also problematic. Finally, non-denaturing conditions used with standard antibody microarrays permit protein complexes, which can produce false positives and false negatives. Changes in the level of an interacting protein may be misinterpreted as alterations in the amount of a target protein or its phosphorylation state. It is critical that leads from both types of microarrays are validated by complementary approaches such as immunoblotting and ELISA. More than a hundred reports have appeared in the scientific literature that have benefited from utilization of antibody and protein lysate microarrays. We have highlighted some of the pioneering works in this field and provided recent examples of their successful deployment as tools for broad-based, targeted proteomics research.     

Protein microarrays for gene expression and antibody screening.

Proteins translate genomic sequence information into function, enabling biological processes. As a complementary approach to gene expression profiling on cDNA microarrays, we have developed a technique for high-throughput gene expression and antibody screening on chip-size protein microarrays. Using a picking/spotting robot equipped with a new transfer stamp, protein solutions were gridded onto polyvinylidene difluoride filters at high density. Specific purified protein was detected on the filters with high sensitivity (250 amol or 10 pg of a test protein). On a microarray made from bacterial lysates of 92 human cDNA clones expressed in a microtiter plate, putative protein expressors could be reliably identified. The rate of false-positive clones, expressing proteins in incorrect reading frames, was low. Product specificity of selected clones was confirmed on identical microarrays using monoclonal antibodies. Cross-reactivities of some antibodies with unrelated proteins imply the use of protein microarrays for antibody specificity screening against whole libraries of proteins. Because this application would not be restricted to antigen-antibody systems, protein microarrays should provide a general resource for high-throughput screens of gene expression and receptor-ligand interactions.     

Assessment and optimisation of normalisation methods for dual-colour antibody microarrays

Background  

Recent advances in antibody microarray technology have made it possible to measure the expression of hundreds of proteins simultaneously in a competitive dual-colour approach similar to dual-colour gene expression microarrays. Thus, the established normalisation methods for gene expression microarrays, e.g. loess regression, can in principle be applied to protein microarrays. However, the typical assumptions of such normalisation methods might be violated due to a bias in the selection of the proteins to be measured. Due to high costs and limited availability of high quality antibodies, the current arrays usually focus on a high proportion of regulated targets. Housekeeping features could be used to circumvent this problem, but they are typically underrepresented on protein arrays. Therefore, it might be beneficial to select invariant features among the features already represented on available arrays for normalisation by a dedicated selection algorithm.     

Protein microarrays: a chance to study microorganisms?

Within the last 5 years, protein microarrays have been developed and applied to multiple approaches: identification of protein–protein interactions or protein–small molecule interactions, cancer profiling, detection of microorganisms and toxins, and identification of antibodies due to allergens, autoantigens, and pathogens. Protein microarrays are small size (typically in the microscopy slide format) planar analytical devices with probes arranged in high density to provide the ability to screen several hundred to thousand known substrates (e.g., proteins, peptides, antibodies) simultaneously. Due to their small size, only minute amounts of spotted probes and analytes (e.g., serum) are needed; this is a particularly important feature, for these are limited or expensive. In this review, different types of protein microarrays are reviewed: protein microarrays (PMAs), with spotted proteins or peptides; antibody microarrays (AMAs), with spotted antibodies or antibody fragments (e.g., scFv); reverse phase protein microarrays (RPMAs), a special form of PMA where crude protein mixtures (e.g., cell lysates, fractions) are spotted; and nonprotein microarrays (NPMAs) where macromolecules other than proteins and nucleic acids (e.g., carbohydrates, monosaccharides, lipopolysaccharides) are spotted. In this study, exemplary experiments for all types of protein arrays are discussed wherever applicable with regard to investigations of microorganisms.     

In situ protein microarrays capable of real-time kinetics analysis based on surface plasmon resonance imaging

In recent years, in situ protein synthesis microarray technologies have enabled protein microarrays to be created on demand just before they are needed. In this paper, we utilized the TUS-TER immobilization technology to allow label-free detection with real-time kinetics of protein–protein interactions using surface plasmon resonance imaging (SPRi). We constructed an expression-ready plasmid DNA with a C-terminal TUS fusion tag to directionally immobilize the in situ synthesized recombinant proteins onto the surface of the biosensor. The expression plasmid was immobilized on the polyethylene imine-modified gold surface, which was then coupled with a cell-free expression system on the flow cell of the SPRi instrument. The expressed TUS fusion proteins bind on the surface via the immobilized TER DNA sequence with high affinity (∼3–7 × 10⁻¹³ M). The expression and immobilization of the recombinant in situ expressed proteins were confirmed by probing with specific antibodies. The present study shows a new low cost method for in situ protein expression microarrays that has the potential to study the kinetics of protein–protein interactions. These protein microarrays can be created on demand without the problems of stability associated with protein arrays used in the drug discovery and biomarker discovery fields.     

Protein microarrays as tools for functional proteomics

Protein microarrays present an innovative and versatile approach to study protein abundance and function at an unprecedented scale. Given the chemical and structural complexity of the proteome, the development of protein microarrays has been challenging. Despite these challenges there has been a marked increase in the use of protein microarrays to map interactions of proteins with various other molecules, and to identify potential disease biomarkers, especially in the area of cancer biology. In this review, we discuss some of the promising advances made in the development and use of protein microarrays.     

Protein microarrays and novel detection platforms

The field of proteomics has undergone rapid advancements over the last decade and protein microarrays have emerged as a promising technological platform for the challenging task of studying complex proteomes. This gel-free approach has found an increasing number of applications due to its ability to rapidly and efficiently study thousands of proteins simultaneously. Different protein microarrays, including capture arrays, reverse-phase arrays, tissue microarrays, lectin microarrays and cell-free expression microarrays, have emerged, which have demonstrated numerous applications for proteomics studies including biomarker discovery, protein interaction studies, enzyme-substrate profiling, immunological profiling and vaccine development, among many others. The need to detect extremely low-abundance proteins in complex mixtures has provided motivation for the development of sensitive, real-time and multiplexed detection platforms. Conventional label-based approaches like fluorescence, chemiluminescence and use of radioactive isotopes have witnessed substantial advancements, with techniques like quantum dots, gold nanoparticles, dye-doped nanoparticles and several bead-based methods now being employed for protein microarray studies. In order to overcome the limitations posed by label-based technologies, several label-free approaches like surface plasmon resonance, carbon nanotubes and nanowires, and microcantilevers, among others, have also advanced in recent years, and these methods detect the query molecule itself. The scope of this article is to outline the protein microarray techniques that are currently being used for analytical and function-based proteomics and to provide a detailed analysis of the key technological advances and applications of various detection systems that are commonly used with microarrays.     

Protein micro- and macroarrays: digitizing the proteome

The early applications of microarrays and detection technologies have been centered on DNA-based applications. The application of array technologies to proteomics is now occurring at a rapid rate. Numerous researchers have begun to develop technologies for the creation of microarrays of protein-based screening tools. The stability of antibody molecules when bound to surfaces has made antibody arrays a starting point for proteomic microarray technology. To minimize disadvantages due to size and availability, some researchers have instead opted for antibody fragments, antibody mimics or phage display technology to create libraries for protein chips. Even further removed from antibodies are libraries of aptamers, which are single-stranded oligonucleotides that express high affinity for protein molecules. A variation on the theme of protein chips arrayed with antibody mimics or other protein capture ligand is that of affinity MS where the protein chips are directly placed in a mass spectrometer for detection. Other approaches include the creation of intact protein microarrays directly on glass slides or chips. Although many of the proteins may likely be denatured, successful screening has been demonstrated. The investigation of protein-protein interactions has formed the basis of a technique called yeast two-hybrid. In this method, yeast "bait" proteins can be probed with other yeast "prey" proteins fused to DNA binding domains. Although the current interpretation of protein arrays emphasizes microarray grids of proteins or ligands on glass slides or chips, 2-D gels are technically macroarrays of authentic proteins. In an innovative departure from the traditional concept of protein chips, some researchers are implementing microfluidic printing of arrayed chemistries on individual protein spots blotted onto membranes. Other researchers are using in-jet printing technology to create protein microarrays on chips. The rapid growth of proteomics and the active climate for new technology is driving a new generation of companies and academic efforts that are developing novel protein microarray techniques for the future.     

Protein microarrays and their applications

In recent years, the importance of proteomic works, such as protein expression, detection and identification, has grown in the fields of proteomic and diagnostic research. This is because complete genome sequences of humans, and other organisms, progress as cellular processing and controlling are performed by proteins as well as DNA or RNA. However, conventional protein analyses are time-consuming; therefore, high throughput protein analysis methods, which allow fast, direct and quantitative detection, are needed. These are so-called protein microarrays or protein chips, which have been developed to fulfill the need for high-throughput protein analyses. Although protein arrays are still in their infancy, technical development in immobilizing proteins in their native conformation on arrays, and the development of more sensitive detection methods, will facilitate the rapid deployment of protein arrays as high-throughput protein assay tools in proteomics and diagnostics. This review summarizes the basic technologies that are needed in the fabrication of protein arrays and their recent applications.     

Protein chip-based microarray profiling of oxidized low density lipoprotein-treated cells

Commercially available high-content Ab380 and extensively validated DLM26 homemade protein microarrays were used to profile the effects of the pro-atherogenic molecule, oxidized low density lipoprotein (OxLDL), on human aortic smooth muscle cells. Protein microarrays detected 298 proteins in cell lysates and 54 of these were differentially regulated. Microarray data were validated by immunoblotting for a selected set of up- and down-regulated proteins. The protein microarray data sets were compared with our recent cDNA microarray-based gene expression results in order to characterize the global effect of OxLDL on smooth muscle cell functions. A group of cell-cell interaction molecules was classified as up-regulated by OxLDL, whereas nucleic acid/protein biosynthesis, structural and humoral response proteins/genes were under-expressed in cells treated by OxLDL. These findings reveal the major pattern of OxLDL-induced effects on the human aortic smooth muscle cells functions and also demonstrate that protein chip-based microarrays could be a useful proteomic tool to profile disease-related states of muscle cells.     

Differential membrane proteomics using 18O-labeling to identify biomarkers for cholangiocarcinoma

Quantitative proteomic methodologies allow profiling of hundreds to thousands of proteins in a high-throughput fashion. This approach is increasingly applied to cancer biomarker discovery to identify proteins that are differentially regulated in cancers. Fractionation of protein samples based on enrichment of cellular subproteomes prior to mass spectrometric analysis can provide increased coverage of certain classes of molecules. We used a membrane protein enrichment strategy coupled with 18O labeling based quantitative proteomics to identify proteins that are highly expressed in cholangiocarcinomas. In addition to identifying several proteins previously known to be overexpressed in cholangiocarcinoma, we discovered a number of molecules that were previously not associated with cholangiocarcinoma. Using immunoblotting and immunohistochemical labeling of tissue microarrays, we validated Golgi membrane protein 1, Annexin IV and Epidermal growth factor receptor pathway substrate 8 (EPS8) as candidate biomarkers for cholangiocarcinomas. Golgi membrane protein 1 was observed to be overexpressed in 89% of cholangiocarcinoma cases analyzed by staining tissue microarrays. In light of recent reports showing that Golgi membrane protein 1 is detectable in serum, further investigation into validation of this protein has the potential to provide a biomarker for early detection of cholangiocarcinomas.     

Peroxisomal matrix protein import

In the past decade, much progress has been made in understanding the mechanisms that govern sorting of proteins to the peroxisomal lumen. This article summarizes the principal features of how peroxisomal matrix enzymes are thought to reach the peroxisome. In addition, it describes recent data that indicate that, in specific pex mutants of the methylotrophic yeast Hansenula polymorpha, defects in matrix protein import can be (partly) rescued by overproduction of the receptor essential for import of these proteins. The implication of these results on the mechanisms of matrix protein import is discussed.     

Recent advances of protein microarrays

Technological innovations and novel applications have greatly advanced the field of protein microarrays. Over the past two years, different types of protein microarrays have been used for serum profiling, protein abundance determinations, and identification of proteins that bind DNA or small compounds. However, considerable development is still required to ensure common quality standards and to establish large content repertoires. Here, we summarize applications available to date and discuss recent technological achievements and efforts on standardization.     

High-throughput fluorescence labelling of full-length cDNA products based on a reconstituted translation system

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyse more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently labelled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labelling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labelled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.