Biosynthetic approach for functional protein microarrays

Protein microarrays have emerged as an indispensable research tool for providing information about protein functions and interactions through high-throughput screening. Traditional methods for immobilizing biomolecules onto solid surfaces have been based on covalent and noncovalent binding, entrapment in semipermeable membranes, microencapsulation, sol gel, and hydrogel methods. Each of these techniques has its own strengths but fails to combine the most important tenets of a functional protein microarray such as covalent attachment, native protein conformation, homogeneity of the protein monolayer, control over active site orientation, and retention of protein activity. Here we present a selective and site-directed covalent immobilization technique for proteins via a benzoxazine ring formation through a Diels-Alder reaction in water and a genetically encoded 3-amino-L-tyrosine (3-NH(2)Tyr) amino acid. Fully functional protein microarrays, with monolayer arrangements and complete control over their orientations, were generated using this strategy.     

Emerging tools for real-time label-free detection of interactions on functional protein microarrays

The availability of extensive genomic information and content has spawned an era of high-throughput screening that is generating large sets of functional genomic data. In particular, the need to understand the biochemical wiring within a cell has introduced novel approaches to map the intricate networks of biological interactions arising from the interactions of proteins. The current technologies for assaying protein interactions--yeast two-hybrid and immunoprecipitation with mass spectrometric detection--have met with considerable success. However, the parallel use of these approaches has identified only a small fraction of physiologically relevant interactions among proteins, neglecting all nonprotein interactions, such as with metabolites, lipids, DNA and small molecules. This highlights the need for further development of proteome scale technologies that enable the study of protein function. Here we discuss recent advances in high-throughput technologies for displaying proteins on functional protein microarrays and the real-time label-free detection of interactions using probes of the local index of refraction, carbon nanotubes and nanowires, or microelectromechanical systems cantilevers. The combination of these technologies will facilitate the large-scale study of protein interactions with proteins as well as with other biomolecules.     

DNA microarrays for functional plant genomics

DNA microarray technology is a key element in today's functional genomics toolbox. The power of the method lies in miniaturization, automation and parallelism permitting large-scale and genome-wide acquisition of quantitative biological information from multiple samples. DNA microarrays are currently fabricated and assayed by two main approaches involving either in situ synthesis of oligonucleotides (`oligonucleotide microarrays') or deposition of pre-synthesized DNA fragments (`cDNA microarrays') on solid surfaces. To date, the main applications of microarrays are in comprehensive, simultaneous gene expression monitoring and in DNA variation analyses for the identification and genotyping of mutations and polymorphisms. Already at a relatively early stage of its application in plant science, microarrays are being utilized to examine a range of biological issues including the circadian clock, plant defence, environmental stress responses, fruit ripening, phytochrome A signalling, seed development and nitrate assimilation. Novel insights are obtained into the molecular mechanisms co-ordinating metabolic pathways, regulatory and signalling networks. Exciting new information will be gained in the years to come not only from genome-wide expression analyses on a few model plant species, but also from extensive studies of less thoroughly studied species on a more limited scale. The value of microarray technology to our understanding of living processes will depend both on the amount of data to be generated and on its clever exploration and integration with other biological knowledge arising from complementary functional genomics tools for `profiling' the genome, proteome, metabolome and phenome.     

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.     

Differential binding studies applying functional protein microarrays and surface plasmon resonance

A variety of different in vivo and in vitro technologies provide comprehensive insights in protein-protein interaction networks. Here we demonstrate a novel approach to analyze, verify and quantify putative interactions between two members of the S100 protein family and 80 recombinant proteins derived from a proteome-wide protein expression library. Surface plasmon resonance (SPR) using Biacore technology and functional protein microarrays were used as two independent methods to study protein-protein interactions. With this combined approach we were able to detect nine calcium-dependent interactions between Arg-Gly-Ser-(RGS)-His6 tagged proteins derived from the library and GST-tagged S100B and S100A6, respectively. For the protein microarray affinity-purified proteins from the expression library were spotted onto modified glass slides and probed with the S100 proteins. SPR experiments were performed in the same setup and in a vice-versa approach reversing analytes and ligands to determine distinct association and dissociation patterns of each positive interaction. Besides already known interaction partners, several novel binders were found independently with both detection methods, albeit analogous immobilization strategies had to be applied in both assays.     

Label-free detection methods for protein microarrays

With the growth of the "-omics" such as functional genomics and proteomics, one of the foremost challenges in biotechnologies has become the development of novel methods to monitor biological process and acquire the information of biomolecular interactions in a systematic manner. To fully understand the roles of newly discovered genes or proteins, it is necessary to elucidate the functions of these molecules in their interaction network. Microarray technology is becoming the method of choice for such a task. Although protein microarray can provide a high throughput analytical platform for protein profiling and protein-protein interaction, most of the current reports are limited to labeled detection using fluorescence or radioisotope techniques. These limitations deflate the potential of the method and prevent the technology from being adapted in a broader range of proteomics applications. In recent years, label-free analytical approaches have gone through intensified development and have been coupled successfully with protein microarray. In many examples of label-free study, the microarray has not only offered the high throughput detection in real time, but also provided kinetics information as well as in situ identification. This article reviews the most significant label-free detection methods for microarray technology, including surface plasmon resonance imaging, atomic force microscope, electrochemical impedance spectroscopy and MS and their applications in proteomics research.     

Functional protein microarrays

Microarrays of immobilized functional proteins have the potential to increase dramatically the throughput of proteomic analysis. Micro-immunoassays, in which biological samples are exposed to arrays of immobilized antibodies, can be used for protein expression profiling. In addition, protein function can be elucidated by performing binding and enzymatic assays on arrays of biologically active proteins.     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

Cell microarrays and functional genomics

With the complete sequencing of the human genome, research priorities have shifted from the identification of genes to the elucidation of their function. Methods currently used by scientists to characterize gene function, such as knock-out mice, are based upon loss of protein function and analysis of the resulting phenotypes to infer a potential role for the protein under scrutiny. Until now, these methods have been successful but time consuming and only a few genes at a time could be analyzed. Cell microarrays allow to simultaneously transfect thousands of different nucleic acid molecules, RNA or DNA, into adherent cells. It is then possible to analyze a large pallet of resulting phenotypes in clusters of transfected cells. We are currently manufacturing cell microarrays with collections of full-length cDNA cloned in expression vectors (gain of function analyses) or siRNA (loss of function studies) to unravel function of genes involved in differentiation and proliferation of human cells. Although there are still some technological difficulties to overcome, the potential for cell microarrays to speed up functional exploration of genomes is very promising.     

Functional protein microarrays: ripe for discovery

The manufacture and use of protein microarrays with correctly folded and functional content presents significant challenges. Despite this, the feasibility and utility of such undertakings are now clear, and exciting progress has recently been demonstrated in the areas of content generation, printing strategies and protein immobilization. More importantly, we are now beginning to enjoy the fruits of these efforts as functional protein microarrays are being increasingly employed for biological discovery purposes. Recent examples of this include the characterization of autoantibody responses, antibody specificity profiling, protein-protein domain interaction profiling and a comprehensive characterization of coiled-coil interactions. The best, however, is yet to come.     

Quantitative protein microarrays for time-resolved measurements of protein phosphorylation

The quantitative analysis of signaling networks requires highly sensitive methods for the time-resolved determination of protein phosphorylation. For this reason, we developed a quantitative protein microarray that monitors the activation of multiple signaling pathways in parallel, and at high temporal resolution. A label-free sandwich approach was combined with near infrared detection, thus permitting the accurate quantification of low-level phosphoproteins in limited biological samples corresponding to less than 50,000 cells, and with a very low standard deviation of approximately 5%. The identification of suitable antibody pairs was facilitated by determining their accuracy and dynamic range using our customized software package Quantpro. Thus, we are providing an important tool to generate quantitative data for systems biology approaches, and to drive innovative diagnostic applications.     

Surface modification for DNA and protein microarrays

Microarrays of biomolecules are emerging as powerful tools for genomics, proteomics, and clinical assays, since they make it possible to screen biologically important binding events in a parallel and high throughput fashion. Because the microarrays are fabricated on a solid support, coating of the surface and immobilization strategy of the biomolecules are major issues for successful microarray fabrication. This review deals with both DNA microarrays and protein microarrays, and focuses on the various modification approaches for the two-dimensional surface materials and three-dimensional ones. In addition, the immobilization strategies including adsorption, covalent attachment, physical entrapment, and affinity attachment of the biomolecules are summarized, and advantage and limitation of representative efforts are discussed.     

A new polymeric coating for protein microarrays

Despite the increasing interest in arraying proteins in a high-density format, several technical issues still impede the development of protein microarray technology. One of the major problems is the availability of substrates that are able to bind native proteins with high density. In this study, we investigated the suitability of a novel surface as a support for protein microarrays. A polymeric glass coating is obtained by physical adsorption of a N,N-dimethylacrylamide (DMA), N,N-acryloyloxysuccinimide (NAS), and [3-(methacryloyl-oxy)propyl]trimethoxysilyl (MAPS) copolymer. The coating procedure provides a fast and inexpensive method of producing hydrophilic functional surfaces. The slide performance was investigated in a protein-protein interaction experiment and in the assessment of rheumatoid factor (RF) in human serum samples. The results demonstrate that the ligands immobilized on the polymeric surface maintain an active conformation and are easily accessible, providing a detection limit of 54amol/spot. Moreover, in the RF assay, after hybridization with the sera, the slides have a low background, leading to a detection limit of 900amol/spot.     

Development of functional gene microarrays for microbial community analysis

Functional gene arrays (FGAs) are a special type of microarrays containing probes for key genes involved in microbial functional processes, such as biogeochemical cycling of carbon, nitrogen, sulfur, phosphorus and metals, virulence and antibiotic resistance, biodegradation of environmental contaminants, and stress responses. FGAs have been demonstrated to be a specific, sensitive, and quantitative tool for rapid analysis of microbial communities from different habitats, such as waters, soils, extreme environments, bioreactors, and human microbiomes. In this review, we first summarize currently reported FGAs, and then focus on the FGA development. We will also discuss several key issues of FGA technology as well as challenges and directions in future FGA development.     

Applications of functional gene microarrays for profiling microbial communities

Functional gene arrays (FGAs) have been considered as a specific, sensitive, quantitative, and high throughput metagenomic tool to detect, monitor and characterize microbial communities. Especially GeoChips, the most comprehensive FGAs have been applied to analyze the functional diversity, composition, structure, and metabolic potential or activity of a variety of microbial communities from different habitats, such as aquatic ecosystems, soils, contaminated sites, extreme environments, and bioreactors. FGAs are able to address fundamental questions related to global change, bioremediation, land use, human health, and ecological theories, and link the microbial community structure to environmental properties and ecosystem functioning. This review focuses on applications of FGA technology for profiling microbial communities, including target preparation, hybridization and data processing, and data analysis. We also discuss challenges and future directions of FGA applications.     

Reverse-phase protein lysate microarrays for cell signaling analysis

'Reverse-phase' protein lysate microarray (RPA) assays use micro-scale, cell lysate dot blots that are printed to a substrate, followed by quantitative immunochemical protein detection, known to be particularly effective across many samples. Large-scale sample collection is a labor-intensive and time-consuming process; the information yielded from RPA assays, however, provides unique opportunities to experimentally interpret theoretical protein networks quantitatively. When specific antibodies are used, RPA can generate 1,000 times more data points using 10,000 times less sample volume than an ordinary western blot, enabling researchers to monitor quantitative proteomic responses for various time-scale and input-dose gradients simultaneously. Hence, the RPA system can be an excellent method for experimental validation of theoretical protein network models. Besides the initial screening of primary antibodies, collection of several hundreds of sample lysates from 1- to 8-h periods can be completed in approximately 10 d; subsequent RPA printing and signal detection steps require an additional 2-3 d.     

Yeast proteomics and protein microarrays

Our understanding of biological processes as well as human diseases has improved greatly thanks to studies on model organisms such as yeast. The power of scientific approaches with yeast lies in its relatively simple genome, its facile classical and molecular genetics, as well as the evolutionary conservation of many basic biological mechanisms. However, even in this simple model organism, systems biology studies, especially proteomic studies had been an intimidating task. During the past decade, powerful high-throughput technologies in proteomic research have been developed for yeast including protein microarray technology. The protein microarray technology allows the interrogation of protein–protein, protein–DNA, protein–small molecule interaction networks as well as post-translational modification networks in a large-scale, high-throughput manner. With this technology, many groundbreaking findings have been established in studies with the budding yeast Saccharomyces cerevisiae, most of which could have been unachievable with traditional approaches. Discovery of these networks has profound impact on explicating biological processes with a proteomic point of view, which may lead to a better understanding of normal biological phenomena as well as various human diseases.     

Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.