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.     

Cell microarrays: an emerging technology for the characterization of antibodies

The possibility to miniaturize and parallelize biological assays has a great impact on the development of biomedical technologies. Here, we describe a simple, miniaturized, and parallelized method employing entire cells from different cell lines displaying a protein of interest on their surface, which were immobilized on a microarray slide. Antibodies were added to these cellular microarrays, and their specific binding to the cell surface proteins was monitored using appropriate fluorescently labeled detection molecules. This new method is applicable for rapidly screening cell surface-specific antibodies with respect to selectivity and cross-reactivity.     

凝胶基片的制备与应用研究

在Bind-Silane^R处理的玻片上交联聚丙烯酰胺凝胶层(15mm×15mm×20μm),戊二醛活化。与末端氨基修饰的寡核苷酸片段共价结合制成芯片。这种芯片能够区分液相中序列不同的Cy3标记的目标核酸。与平面基片相比,凝胶基片具有背景低、固定探针量高、杂交时间短的优点。将细胞因子IL-4、IL-5、IL-6、IL-7、ANG、I-309和VEGF的单克隆抗体加样于凝胶基片上制成蛋白质芯片,对乳腺癌患者和正常人的血清进行检测,发现乳腺癌患者细胞因子IL-4、IL-5、I-309和VEGF的表达量高于正常人的表达量,对临床诊断具有重要的参考意义。     

High-throughput protein arrays: prospects for molecular diagnostics

High-throughput protein arrays allow the miniaturized and parallel analysis of large numbers of diagnostic markers in complex samples. Using automated colony picking and gridding, cDNA or antibody libraries can be expressed and screened as clone arrays. Protein microarrays are constructed from recombinantly expressed, purified, and yet functional proteins, entailing a range of optimized expression systems. Antibody microarrays are becoming a robust format for expression profiling of whole genomes. Alternative systems, such as aptamer, PROfusion, nano- and microfluidic arrays are all at proof-of-concept stage. Differential protein profiles have been used as molecular diagnostics for cancer and autoimmune diseases and might ultimately be applied to screening of high-risk and general populations.     

Chemoselective surface immobilization of proteins through a cleavable peptide

Surface immobilization of biomolecules is a fundamental step in several experimental techniques such as surface plasmon resonance analysis and microarrays. Oxime ligation allows reaching chemoselective protein immobilization with the retention of native-like conformation by proteins. Beside the need for chemoselective ligation of molecules to surface/particle, equally important is the controlled release of the immobilized molecules, even after a specific binding event. For this purpose, we have designed and assessed in an SPR experiment a peptide linker able to (i) anchor a given protein (enzymes, receptors, or antibodies) to a surface in a precise orientation and (ii) release the immobilized protein after selective enzymatic cleavage. These results open up the possibility to anchor to a surface a protein probe leaving bioactive sites free for interaction with substrates, ligands, antigens, or drugs and successively remove the probe-ligand complex by enzymatic cleavage. This peptide linker can be considered both an improvement of SPR analysis for macromolecular interaction and a novel strategy for drug delivery and biomaterial developments.     

Strategy for surveying the proteome using affinity proteomics and mass spectrometry

Antibody‐based microarrays is a rapidly evolving technology that has gone from the first proof‐of‐concept studies to more demanding proteome profiling applications, during the last years. Miniaturized microarrays can be printed with large number of antibodies harbouring predetermined specificities, capable of targeting high‐ as well as low‐abundant analytes in complex, nonfractionated proteomes. Consequently, the resolution of such proteome profiling efforts correlate directly to the number of antibodies included, which today is a key limiting factor. To overcome this bottleneck and to be able to perform in‐depth global proteome surveys, we propose to interface affinity proteomics with MS‐based read‐out, as outlined in this technical perspective. Briefly, we have defined a range of peptide motifs, each motif being present in 5–100 different proteins. In this manner, 100 antibodies, binding 100 different motifs commonly distributed among different proteins, would potentially target a protein cluster of 10⁴ individual molecules, i.e. around 50% of the nonredundant human proteome. Notably, these motif‐specific antibodies would be directly applicable to any proteome in a specie independent manner and not biased towards abundant proteins or certain protein classes. The biological sample is digested, exposed to these immobilized antibodies, whereby motif‐containing peptides are specifically captured, enriched and subsequently detected and identified using MS.     

Protein and small molecule microarrays: powerful tools for high-throughput proteomics

Advances in genomics and proteomics have opened up new possibilities for the rapid functional assignment and global characterization of proteins. Large-scale studies have accelerated this effort by using tools and strategies that enable highly parallel analysis of huge repertoires of biomolecules. Organized assortments of molecules on arrays have furnished a robust platform for rapid screening, lead discovery and molecular characterization. The essential advantage of microarray technology is attributed to the massive throughput attainable, coupled with a highly miniaturized platform--potentially driving discovery both as an analytical and diagnostic tool. The scope of microarrays has in recent years expanded impressively. Virtually every biological component--from diverse small molecules and macromolecules (such as DNA and proteins) to entire living cells--has been harnessed on microarrays in attempts to dissect the bewildering complexity of life. Herein we highlight strategies that address challenges in proteomics using microarrays of immobilized proteins and small molecules. Of specific interest are the techniques involved in stably immobilizing proteins and chemical libraries on slide surfaces as well as novel strategies developed to profile activities of proteins on arrays. As a rapidly maturing technology, microarrays pave the way forward in high-throughput proteomic exploration.     

Design of high-density antibody microarrays for disease proteomics: Key technological issues

Antibody-based microarray is a novel proteomic technology setting a new standard for molecular profiling of non-fractionated complex proteomes. The first generation of antibody microarrays has already demonstrated its potential for generating detailed protein expression profiles, or protein atlases, of human body fluids in health and disease, paving the way for new discoveries within the field of disease proteomics. The process of designing highly miniaturized, high-density and high-performing antibody microarray set-ups have, however, proven to be challenging. In this mini-review we discuss key technological issues that must be addressed in a cross-disciplinary manner before true global proteome analysis can be performed using antibody microarrays.     

Diagnostic and analytical applications of protein microarrays

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein–protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.     

Diagnostic and analytical applications of protein microarrays

DNA microarrays have changed the field of biomedical sciences over the past 10 years. For several reasons, antibody and other protein microarrays have not developed at the same rate. However, protein and antibody arrays have emerged as a powerful tool to complement DNA microarrays during the past 5 years. A genome-scale protein microarray has been demonstrated for identifying protein-protein interactions as well as for rapid identification of protein binding to a particular drug. Furthermore, protein microarrays have been shown as an efficient tool in cancer profiling, detection of bacteria and toxins, identification of allergen reactivity and autoantibodies. They have also demonstrated the ability to measure the absolute concentration of small molecules. Besides their capacity for parallel diagnostics, microarrays can be more sensitive than traditional methods such as enzyme-linked immunosorbent assay, mass spectrometry or high-performance liquid chromatography-based assays. However, for protein and antibody arrays to be successfully introduced into diagnostics, the biochemistry of immunomicroarrays must be better characterized and simplified, they must be validated in a clinical setting and be amenable to automation or integrated into easy-to-use systems, such as micrototal analysis systems or point-of-care devices.     

Protein microarrays on hybrid polymeric thin films prepared by self-assembly of polyelectrolytes for multiple-protein immunoassays

We report here the development and characterization of protein microarrays fabricated on nanoengineered 3-D polyelectrolyte thin films (PET) deposited on glass slide by consecutive adsorption of polyelectrolytes via self-assembly technique. Antibodies or antigens were immobilized in the PET-coated glass slides by electrostatic adsorption and entrapment of porous structure of the 3-D polymer film and thus establishing a platform for parallel analysis. Both antigen and antibody microarrays were fabricated on the PET-coated slides, and direct and indirect immunoassays on protein microarrays for multiple-analyte detection were demonstrated. Microarrays produced on these PET-coated slides have consistent spot morphology and provide performance features needed for proteomic analysis. The protein microarrays on the PET films provide LOD as low as 6 pg/mL and dynamic ranges up to three orders of magnitude, which are wider than the protein microarrays fabricated on aldehyde and poly-L-lysine functionalized slides. The PET films constructed by self-assembly technique in aqueous solution is green chemistry based, cost-effective method to generate 3-D thin film coatings on glass surface, and the coated slide is well suited for immobilizing many types of biological molecules so that a wide variety of microarray formats can be developed on this type of slide.     

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.     

Protein microarrays to study carbohydrate-recognition events

In order to expand areas in which protein microarrays can be used to solve important biological problems, we have investigated ways in which the technique can be employed for functional glycomics. Initially, our protein microarrays were used for the rapid identification of carbohydrate-binding proteins using trifunctional carbohydrate probes and fluorescent dye-labeled polysaccharides. Glycan probes were selectively bound to the corresponding lectins immobilized on the solid surface. In addition, these microarrays were also employed for profiling of carbohydrates on Jurkat T-cell surfaces. These cells adhered to ConA, RCA(120), SNA and WGA, indicating expression of alpha-Man, Gal, NeuNAcalpha2,6Gal and GlcNAc residues on their surfaces. Furthermore, we determined binding affinities between WGA and carbohydrates by measuring IC(50) values of GlcNAc that inhibited 50% of trivalent GlcNAc binding to WGA immobilized on the solid surface. All the experiments show that protein microarrays can be used to study carbohydrate-recognition events in the field of glycomics.     

DNA microarrays with unimolecular hairpin double-stranded DNA probes: fabrication and exploration of sequence-specific DNA/protein interactions

We have fabricated double-stranded DNA (dsDNA) microarrays containing unimolecular hairpin dsDNA probes immobilized on glass slides. The unimolecular hairpin dsDNA microarrays were manufactured by four steps: Firstly, synthesizing single-stranded DNA (ssDNA) oligonucleotides with two reverse-complementary sequences at 3' hydroxyl end and an overhang sequence at 5' amino end. Secondly, microspotting ssDNA on glutaraldehyde-derived glass slide to form ssDNA microarrays. Thirdly, annealing two reverse-complementary sequences to form hairpin primer at 3' end of immobilized ssDNA and thus to create partial-dsDNA microarray. Fourthly, enzymatically extending hairpin primer to convert partial-dsDNA microarrays into complete-dsDNA microarray. The excellent efficiency and high accuracy of the enzymatic synthesis were demonstrated by incorporation of fluorescently labeled dUTPs in Klenow extension and digestion of dsDNA microarrays with restriction endonuclease. The accessibility and specificity of the DNA-binding proteins binding to dsDNA microarrays were verified by binding Cy3-labeled NF-kappaB to dsDNA microarrays. The dsDNA microarrays have great potential to provide a high-throughput platform for investigation of sequence-specific DNA/protein interactions involved in gene expression regulation, restriction and so on.     

Thiol-directed immobilization of recombinant IgG-binding receptors

A genetic engineering approach is described for directed immobilization of IgG-binding receptors to a thiol-containing matrix using a single cysteine residue. The cysteine residue is introduced into the C-terminal part of receptors based on staphylococcal protein A. Receptors containing one, two or five IgG-binding domains were produced in Escherichia coli and subsequently immobilized on thiopropyl-Sepharose. A high amount, 5 mumol/ml gel (45 mg/ml), of recombinant receptor could be rapidly immobilized to the solid support and both the gel and the immobilized receptor could be regenerated by reduction and oxidation reactions. The gel was used for affinity purification of human IgG and analysis of IgG-binding capacity at different amounts of immobilized recombinant protein show the same maximal IgG-binding capacity (20-25 mg/ml) for all three immobilized receptors. However, at low substitution grade of receptors, the immobilized receptor molecules were shown to bind one (Z-Cys) and two (ZZ-Cys) IgG molecules, respectively. These results demonstrate that the immobilized protein molecules are in a functionally active form and that a two-domain receptor can bind two molecules of IgG without steric hindrance. Interestingly, the five-domain receptor (ZV-Cys), with a structure similar to native protein A, can only bind approximately two IgG molecules, despite the five-domain structure of the molecule.     

Quantitative assessment of factors affecting the sensitivity of a competitive immunomicroarray for pesticide detection

Analytical protein microarrays offering highly parallel analysis can become an invaluable tool for a wide range of immunodiagnostic applications. Here we describe factors that influence the sensitivity of a competitive immunomicroarray that quantifies small molecules; in this case, the pesticides dichlobenil metabolite 2,6-dichlorobenzamide (BAM) and atrazine. Free pesticide concentrations in solution are quantified by the competitive binding of fluorescence-conjugated monoclonal antibodies to either surface-immobilized pesticide hapten-protein conjugates or pesticides in solution. We investigated the influence of antibody labeling techniques, microarray substrates, and spotting and incubation buffers. The results showed that microarrays immobilized on EasySpot or in-house fabricated agarose substrates printed with Genetix Amine Spotting Solution resulted in optimum results when the arrays were incubated with the sample/antibodies diluted in a Tris buffer supplemented with 0.05% each bovine serum albumin (BSA) and Tween 20. Furthermore, the application of directly labeled primary antibodies allowed for better sensitivity compared to secondary polyclonal antibody quantification.     

Cartilage link protein interacts with neurocan, which shows hyaluronan binding characteristics different from CD44 and TSG-6

The interaction of neurocan with hyaluronan was qualitatively characterized with alkaline phosphatase fusion proteins secreted by mammalian cells. The wild type neurocan hyaluronan binding domain fused to alkaline phosphatase bound to immobilized hyaluronan under physiological as well as moderately hypertonic conditions, whereas its ability to bind to immobilized chondroitin sulfate dropped rapidly with increasing salt concentration. Strong hyaluronan binding ability was still evident when in both link modules within the hyaluronan binding domain a basic amino acid was mutated, which is well conserved among link modules of hyaluronan binding proteins. A strong enhancement of the binding of neurocan to immobilized hyaluronan was observed after preincubation of the immobilized hyaluronan with cartilage link protein. Moreover, this preincubation mediated also the binding of a fusion protein representing only the immunoglobulin module of neurocan linked to alkaline phosphatase, which showed no binding to immobilized hyaluronan alone. The interaction of the neurocan immunoglobulin module with link protein could also be shown by overlay blot analysis. These observations suggest that the hyaluronan binding characteristics of paired link modules are different from those of single link modules, and that the reported temporal co-expression of cartilage link protein and of neurocan in developing brain implicates the possibility of a cooperative function of these molecules.     

Enzymatic activity on a chip: the critical role of protein orientation

We compare the catalytic activities of enzymes immobilized on silicon surfaces with and without orientation. While oriented sulfotransferases selectively immobilized on an otherwise zero-background surface via 6xHis tags faithfully reflect activities of solution phase enzymes, those with random orientation on the surface do not. This finding demonstrates that controlling the orientation of immobilized protein molecules and designing an ideal local chemical environment on the solid surface are both essential if protein microarrays are to be used as quantitative tools in biomedical research.