Improving protein array performance: focus on washing and storage conditions

For protein microarrays, maintaining protein stability during the slide processing steps of washing, drying, and storage is of major concern. Although several studies have focused on the stability of immobilized antibodies in antibody microarrays, studies on protein-protein interaction arrays and enzyme arrays are lacking. In this paper we used five bait-prey protein interaction pairs and three enzymes to optimize the washing, drying, and storage conditions for protein arrays. The protein arrays for the study were fabricated by combining HaloTag technology and cell-free protein expression. The HaloTag technology, in combination with cell-free expression, allowed rapid expression and immobilization of fusion proteins on hydrogel-coated glass slides directly from cell extracts without any prior purification. Experimental results indicate enzyme captured on glass slides undergoes significant loss of activity when washed and spin-dried using only phosphate buffer, as is typically done with antibody arrays. The impact of washing and spin-drying in phosphate buffer on protein-protein interaction arrays was minimal. However, addition of 5% glycerol to the wash buffer helps retain enzyme activity during washing and drying. We observed significant loss of enzyme activity when slides were stored dry at 4 degrees C, however immobilized enzymes remained active for 30 days when stored at -20 degrees C in 50% glycerol. We also found that cell-free extract containing HaloTag-fused enzymes could undergo multiple freeze/thaw cycles without any adverse impact on enzyme activity. The findings indicate that for large ongoing studies, proteins of interest expressed in cell-free extract can be stored at -70 degrees C and repeatedly used to print small batches of protein array slides to be used over a few weeks.     

Antibody array analysis of labelled proteomes: how should we control specificity?

Researchers who use protein binders in multiplexed assays can be divided into two camps. One believes that arrays with proteome-wide coverage will become a reality once we have developed binders for all proteins. The sceptics claim that detection with immobilized protein binders and sample labelling will not provide the required specificity. In this article, we review the evidence showing that antibody array analysis of labelled samples can provide meaningful data and discuss the issues raised by the sceptics. We argue that direct the evidence for monospecificity has yet to be published. This will require assays designed to resolve the proteins captured by each binder. One option is to combine array measurement with protein separation. We have developed an assay where labelled sample proteins are separated by size exclusion chromatography (SEC) before contact with microsphere-based arrays (Size-MAP; size exclusion chromatography-resolved microsphere-based affinity proteomics). The effect is an 'antibody array Western blot' where reactivity of immobilized binders is resolved against the size of the proteins in the sample. We show that Size-MAP is useful to discriminate monospecific- and polyreactive antibodies and for automatic detection of reacting with the same target. The possibility to test specificity directly in array-based measurement should be useful to select the best binders and to determine whether the DNA microarray for the proteome is a realistic goal or not.     

蛋白质微阵列检测抗原-抗体相互作用

为了制备蛋白质微阵列和研究芯片表面抗原-抗体的相互作用,研究了如何在玻片表面固化蛋白质和用荧光染料（Cy3,Cy5）对蛋白质进行标记.结果表明,在醛基修饰的玻璃表面,通过共价偶联的方法将抗原或抗体固定到芯片表面,能使二者保持其特异性结合能力.同时,荧光标记后的抗原或抗体仍然具有特异性结合能力.蛋白质微阵列是通过机械手在玻片表面排阵制作的.芯片上的荧光信号获取采用了激光共焦荧光扫描系统.用不同浓度的抗原探针阵列,对其相应的抗体靶分子的特异性结合进行了分析和研究.此外,还通过在玻片表面固定兔IgG和固定鼠IgG,对羊抗兔和羊抗鼠抗体与其相应抗原的特异性相互作用进行了检测.     

Antibodies immobilized as arrays to profile protein post-translational modifications in mammalian cells

Previously, we demonstrated that antibodies printed on a solid support were able to detect protein-protein interaction in mammalian cells. Here we further developed the antibody array system for detecting proteins with various post-translational modifications in mammalian cells. In this novel approach, immunoprecipitated proteins were labeled with fluorescent dye followed by incubation over antibody arrays. Targeted proteins, captured by the antibodies immobilized on PVDF membrane or glass slide, were detected by means of near infrared fluorescent scanner or fluorescent microscopy. To demonstrate the application of the antibody arrays in protein post-translational modifications, we profiled protein tyrosine phosphorylation, ubiquitination, and acetylation in mammalian cells under different conditions. Our results indicate that antibody array technology can provide a powerful means of profiling a large number of proteins with different post-translational modifications in cells.     

Using a ubiquitin ligase as an unfolded protein sensor

A significant fraction of all proteins are misfolded and must be degraded. The ubiquitin-proteasome pathway provides an essential protein quality control function necessary for normal cellular homeostasis. Substrate specificity is mediated by proteins called ubiquitin ligases. In the endoplasmic reticulum (ER) a specialized pathway, the endoplasmic reticulum associated degradation (ERAD) pathway provides means to eliminate misfolded proteins from the ER. One marker used by the ER to identify misfolded glycoproteins is the presence of a high-mannose (Man5-8GlcNAc2) glycan. Recently, FBXO2 was shown to bind high mannose glycans and participate in ERAD. Using glycan arrays, immobilized glycoprotein pulldowns, and glycan competition assays we demonstrate that FBXO2 preferentially binds unfolded glycoproteins. Using recombinant, bacterially expressed GST-FBXO2 as an unfolded protein sensor we demonstrate it can be used to monitor increases in misfolded glycoproteins after physiological or pharmaceutical stressors.     

Peptide arrays with designed secondary structures for protein characterization using fluorescent fingerprint patterns

To realize a practical high-throughput protein-detection system, novel peptide arrays have been constructed using designed peptide libraries with loop, alpha-helix, or beta-strand structures. Here, we describe the overview of the reported designed peptide arrays with loop and alpha-helix structures and the new results of those with beta-strand structures. Initially, several model peptides known to interact with model structured proteins were selected to establish the present strategy for high-throughput detection of proteins. The fluorescent probes and suitable scaffolds of peptides were examined for the effective detection of proteins. The detection methods were established in solution and in an immobilized manner using the model systems. In the case of alpha-helix peptide, the response of a peptide with fluorescent resonance energy transfer between two probes at both termini was several times higher than that of a peptide with a single probe. In the cases of peptides with other structures, however, proteins were effectively detectable even by the fluorescent change of one probe. Furthermore, structurally focused libraries consisting of a total of ca. 250 different peptides based on the model peptides with secondary and/or tertiary structures were constructed with systematic replacement of residues. Using these libraries, various proteins were characterized effectively to give their own fluorescent "protein fingerprint" patterns. The resulting protein fingerprints correlated with the recognition properties of the proteins. These studies demonstrate that arrays with peptide libraries based on designed structures can be promising tools for detecting the target proteins. Designed synthetic peptides play roles as the capturing agents to be developed for practical protein chips.     

Application of sector protein microarrays to clinical samples

Many protein functions are conferred by posttranslational modifications, which allow proteins to perform specific cellular tasks. Protein microarrays enable specific detection of posttranslational modifications not attainable by gene arrays. Reverse-phase protein microarrays have been widely adopted for use with clinical biopsy specimens because they have many advantages including highly reproducible printing of cellular lysates onto array surfaces, buit-in dilution curves, and direct detection using one antibody per analyte. This results in high-sensitivity, broad dynamic range, and favorable precision. Reverse-phase arrays have been restricted to a one slide/one antibody format. Although this is suitable for analyzing treatment effects over populations of samples, it is not well suited to individual patient assessments. One means of reaching this goal is the sector array format. Through the sector array, multiple antibody probes can be multiplexed on a single slide containing replicate immobilized aliquots from one patient. Thus, on one slide, a complete set of analytes can be characterized and used to support a therapy decision. This article describes a method for constructing sector arrays and demonstrates feasibility and adequate sensitivity applied to apoptosis related pathways.     

Fabrication of an antibody microwell array with self-adhering antibody binding protein

One of the promising methods of preparing antibody arrays is immobilizing antibodies with protein A or protein G, each of which binds specifically to the heavy chain constant (Fc) region of immunoglobulin G (IgG). In this system, antibody immobilization efficiency depends on the number of active Fc binding proteins that need to be immobilized on the surface. Here we have designed and constructed an Fc binding protein with a self-adhering ability that can be immobilized on the hydrophobic surface by simple adsorption. It consists of an Fc binding domain of protein G (G3) and hydrophobic domain of elastin (E72). Direct observation revealed its self-adhering ability on the hydrophobic surface. The enzyme-linked immunosorbent assay (ELISA) showed that it retained antibody binding ability on the surface. The antibody array model was prepared on a hydrophobic microwell glass slide with E72G3, which specifically detect the antigen with a sevenfold greater sensitivity than the G3-treated slide. These results suggest that the E72G3 is useful for simple and effective immobilization of antibodies and can be used to fabricate any immuno devices.     

Peptide arrays: towards routine implementation

Peptide arrays have attracted wide interest as tools for discovering biochemical interactions. Because many protein binding and enzyme activities are directed towards peptides, the preparation of arrays having hundreds to thousands of immobilized peptides offers an unprecedented opportunity for identifying interactions of proteins. This short opinion reviews recent progress in the preparation of peptide arrays and their use in the characterization of biomolecular interactions and the discovery of new reagents for biological researches. This body of work establishes the feasibility of this technology and suggests that it will find much wider use in research groups.     

The ProteinChip Biomarker System from Ciphergen Biosystems: a novel proteomics platform for rapid biomarker discovery and validation

Protein biochips are emerging in two distinct formats. The first involves high-density immobilized arrays of recognition molecules (e.g. antibodies), where target binding is monitored indirectly (e.g. via fluorescence). This technology is in its infancy, being limited by the availability of suitable binding molecules that can cope effectively with protein diversity. The second format involves the capture of proteins by biochemical or intermolecular interaction, coupled with direct detection by MS. This technology is available as the ProteinChip Biomarker System. ProteinChip technology uses surface-enhanced laser desorption/ionization processes to analyse proteins directly from biological samples. Chromatographic surfaces are placed on to ProteinChip Arrays and used to capture subclasses of proteins, dependent on their physical properties. Time-of-flight MS then assigns native molecular masses to the captured proteins. Reproducible protein profiles can be generated from crude biological fluids (e.g. cell lysates, urine or serum). The technology is being applied to a wide range of disciplines, from plant sciences to cancer research, and will be reviewed here.     

Preparation of DNA and protein micro arrays on glass slides coated with an agarose film

A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries. An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single reaction with minimal amounts of the binding reagents.     

UV immobilized phospholipid bilayers

Dinitrophenyl phosphotidylethanolamine-containing bilayers have been immobilized on carbon-shadowed support films by UV irradiation of the first monolayer transferred to the support film. The immobilized bilayer is capable of allowing bound protein (anti-DNP antibody) to organize into 2-D arrays in the presence of organic solvents such as acetonitrile and dilute concentrations of detergents such as beta-octyl glucoside. The ability of the bilayers to remain attached to supports under various conditions that include organic solvents and detergents as well as divalent ions is of potential interest in the study of protein crystallization and particularly in the study of membrane proteins.     

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.     

News in Brief

Protein microarrays are versatile tools for parallel, miniaturized screening of binding events involving large numbers of immobilized proteins in a time- and cost-effective manner. They are increasingly applied for high-throughput protein analyses in many research areas, such as protein interactions, expression profiling and target discovery. While conventionally made by the spotting of purified proteins, recent advances in technology have made it possible to produce protein microarrays through in situ cell-free synthesis directly from corresponding DNA arrays. This article reviews recent developments in the generation of protein microarrays and their applications in proteomics and diagnostics.     

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.     

Immunostaining with dissociable antibody microarrays

The availability of a large number of biological materials such as cDNA, antibodies, recombinant proteins, and tissues has promoted the development of microarray technologies that make use of these materials in high-throughput screening assays. However, because microarray technologies have been less successful in examining proteins than DNA and mRNA, there is a need for improved protein microarray systems. To address this need, we developed an antibody microarray-based immunostaining method that can analyze the properties of a large number of proteins simultaneously. In this method, antibodies are arrayed and immobilized on a solid support and cells bearing antigens of interest are attached to a second support. Apposition of the two supports allows the antibodies to dissociate from the array support and bind to the cellular antigens. After separation of the supports, antigen-bound antibodies can be detected by standard secondary antibody techniques. These "dissociable" antibody arrays were used to detect both the expression and subcellular localization of a large number of specific proteins in various cultured cell types.     

Self-assembling protein arrays using electronic semiconductor microchips and in vitro translation

Protein arrays will greatly accelerate research and development in medical and biological sciences. We have used cell-free protein biosynthesis and a parallel immobilization strategy for producing protein biochips. We demonstrate a model two-protein microarray using luciferase and green fluorescent protein, both expressed in a cell-free system and specifically immobilized on CombiMatrix semiconductor oligonucleotide microarrays. This demonstration provides evidence for the appropriate folding, activity, robust presentation, and efficient flexible detection of proteins on the microscale.     

Proteomic analysis of in vivo phosphorylated synaptic proteins

In the nervous system, protein phosphorylation is an essential feature of synaptic function. Although protein phosphorylation is known to be important for many synaptic processes and in disease, little is known about global phosphorylation of synaptic proteins. Heterogeneity and low abundance make protein phosphorylation analysis difficult, particularly for mammalian tissue samples. Using a new approach, combining both protein and peptide immobilized metal affinity chromatography and mass spectrometry data acquisition strategies, we have produced the first large scale map of the mouse synapse phosphoproteome. We report over 650 phosphorylation events corresponding to 331 sites (289 have been unambiguously assigned), 92% of which are novel. These represent 79 proteins, half of which are novel phosphoproteins, and include several highly phosphorylated proteins such as MAP1B (33 sites) and Bassoon (30 sites). An additional 149 candidate phosphoproteins were identified by profiling the composition of the protein immobilized metal affinity chromatography enrichment. All major synaptic protein classes were observed, including components of important pre- and postsynaptic complexes as well as low abundance signaling proteins. Bioinformatic and in vitro phosphorylation assays of peptide arrays suggest that a small number of kinases phosphorylate many proteins and that each substrate is phosphorylated by many kinases. These data substantially increase existing knowledge of synapse protein phosphorylation and support a model where the synapse phosphoproteome is functionally organized into a highly interconnected signaling network.     

Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation

Methods to assess the quality and performance of protein microarrays fabricated from undefined protein content are required to elucidate slide-to-slide variability and interpolate resulting signal intensity values after an interaction assay. We therefore developed several simple total- and posttranslational modification-specific, on-chip staining methods to quantitatively assess the quality of gel element protein arrays manufactured with whole-cell lysate in vitro protein fractions derived from two-dimensional liquid-phase fractionation (PF2D) technology. A linear dynamic range of at least 3 logs was observed for protein stains and immobilized protein content, with a lower limit of detection at 8 pg of protein per gel element with Deep Purple protein stain and a field-portable microarray imager. Data demonstrate the successful isolation, separation, transfer, and immobilization of putative transmembrane proteins from Yersinia pestis KIM D27 with the combined PF2D and gel element array method. Internal bovine serum albumin standard curves provided a method to assess on-chip PF2D transfer and quantify total protein immobilized per gel element. The basic PF2D array fabrication and quality assurance/quality control methods described here therefore provide a standard operating procedure and basis for developing whole-proteome arrays for interrogating host-pathogen interactions, independent of sequenced genomes, affinity tags, or a priori knowledge of target cell composition.     

Protein and peptide arrays: recent trends and new directions

Microarrays of proteins and peptides make it possible the screening of thousands of binding events in a parallel and high throughput fashion; therefore they are emerging as a powerful tool for proteomics and clinical assays. The complex nature of Proteome, the wide dynamic range of protein concentration in real samples and the critical role of immobilized protein orientation must be taken into account to maximize the utility of protein microarrays. Immobilization strategy and designing of an ideal local chemical environment on the solid surface are both essential for the success of a protein microarray experiment. This review article will focus on protein and peptide arrays highlighting their technical challenges and presenting new directions by means of a set of selected recent applications.