Improving the intein-mediated, site-specific protein biotinylation strategies both in vitro and in vivo

One of the critical issues in the generation of a protein microarray lies in the choice of immobilization strategies, which ensure proteins are adhered to the glass surface while properly retaining their native biological activities. We previously developed intein-mediated strategies for protein biotinylation and site-specific protein microarray generation. Herein, we report new findings of these strategies, which improve the biotinylation efficiency of proteins by up to 10-folds.     

Engineering of a phosphorylatable tag for specific protein binding on zirconium phosphonate based microarrays

A phosphorylatable tag was designed and fused at the C-terminal end of proteins, which allowed efficient and oriented immobilization of capture proteins on glass substrates coated with a zirconium phosphonate monolayer. The concept is demonstrated using Nanofitin directed against lysozyme. This peptide tag (DSDSSSEDE) contains four serines in an acidic environment, which favored its in vitro phosphorylation by casein kinase II. The resulting phosphate cluster at the C-terminal end of the protein provided a specific, irreversible, and multipoint attachment to the zirconium surface. In a microarray format, the high surface coverage led to high fluorescence signal after incubation with Alexa Fluor 647 labeled lysozyme. The detection sensitivity of the microarray for the labeled target was below 50 pM, owing to the exceptionally low background staining, which resulted in high fluorescence signal to noise ratios. The performance of this new anchoring strategy using a zirconium phosphonate modified surface compares favorably with that of other types of microarray substrates, such as nitrocellulose-based or epoxide slides, which bind proteins in a nonoriented way.     

Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.     

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.     

Strategies for site-specific protein biotinylation using in vitro, in vivo and cell-free systems: toward functional protein arrays

This protocol details methodologies for the site-specific biotinylation of proteins using in vitro, in vivo and cell-free systems for the purpose of fabricating functional protein arrays. Biotinylation of recombinant proteins, in vitro as well as in vivo, relies on the chemoselective reaction between cysteine-biotin and a reactive thioester group at the C-terminus of a protein generated via intein-mediated cleavage. The cell-free system utilizes low concentrations of biotin-conjugated puromycin. Unlike other approaches that require tedious and costly downstream steps of protein purification, C-terminal biotinylated proteins can be captured directly onto avidin-functionalized slides from a mixture of other cellular proteins to generate the corresponding protein array. These methods were designed to maintain the integrity and activity of proteins in a microarray format, which potentially allows simultaneous functional assays of thousands of proteins. Assuming that the target proteins have been cloned into the expression vector, transformation of bacterial strain and growth of starter culture would take approximately 2 days. Expression and in vitro protein purification and biotinylation will take approximately 3 days whereas the in vivo method would take approximately 2 days. The cell-free protein biotinylation strategy requires only 6-8 h.     

Antibody microarray profiling of human prostate cancer sera: antibody screening and identification of potential biomarkers

We developed a practical strategy for serum protein profiling using antibody microarrays and applied the method to the identification of potential biomarkers in prostate cancer serum. Protein abundances from 33 prostate cancer and 20 control serum samples were compared to abundances from a common reference pool using a two-color fluorescence assay. Robotically spotted microarrays containing 184 unique antibodies were prepared on two different substrates: polyacrylamide based hydrogels on glass and poly-1-lysine coated glass with a photoreactive cross-linking layer. The hydrogel substrate yielded an average six-fold higher signal-to-noise ratio than the other substrate, and detection of protein binding was possible from a greater number of antibodies using the hydrogels. A statistical filter based on the correlation of data from "reverse-labeled" experiment sets accurately predicted the agreement between the microarray measurements and enzyme-linked immunosorbent assay measurements, showing that this parameter can serve to screen for antibodies that are functional on microarrays. Having defined a set of reliable microarray measurements, we identified five proteins (von Willebrand Factor, immunoglobulinM, Alpha1-antichymotrypsin, Villin and immunoglobulinG) that had significantly different levels between the prostate cancer samples and the controls. These developments enable the immediate use of high-density antibody and protein microarrays in biomarker discovery studies.     

Microcontact printing of biotin for selective immobilization of streptavidin-fused proteins and SPR analysis

In this study, a simple procedure is described for patterning biotin on a glass substrate and then selectively immobilizing proteins of interest onto the biotin-patterned surface. Microcontact printing (μCP) was used to generate the micropattern of biotin and to demonstrate the selective immobilization of proteins by using enhanced green fluorescent protein (EGFP) as a model protein, of which the C-terminus was fused to a core streptavidin (cSA) gene ofStreptomyces avidinii. Confocal fluorescence microscopy was used to visualize the pattern of the immobilized protein (EGFP-cSA), and surface plasmon resonance was used to characterize biological activity of the immobilized EGFP-cSA. The results suggest that this strategy, which consists of a combination of μμCP and cSA-fused proteins, is an effective way for fabricating biologically active substrates that are suitable for a wide variety of applications, one such being the use in protein-protein assays. These authors equally contributed to this study     

Lithographic techniques and surface chemistries for the fabrication of PEG-passivated protein microarrays

This article presents a new technique to fabricate patterns of functional molecules surrounded by a coating of the inert poly(ethylene glycol) (PEG) on glass slides for applications in protein microarray technology. The chief advantages of this technique are that it is based entirely on standard lithography processes, makes use of glass slides employing surface chemistries that are standard in the microarray community, and has the potential to massively scale up the density of microarray spots. It is shown that proteins and antibodies can be made to self-assemble on the functional patterns in a microarray format, with the PEG coating acting as an effective passivating agent to prevent non-specific protein adsorption. Various standard surface chemistries such as aldehyde, epoxy and amine are explored for the functional layer, and it is conclusively demonstrated that only an amine-terminated surface satisfies all the process constraints imposed by the lithography process sequence. The effectiveness of this microarray technology is demonstrated by patterning fluorescent streptavidin and a fluorescent secondary antibody using the well-known and highly specific interaction between biotin and streptavidin.     

Cell adhesion profiling using extracellular matrix protein microarrays

We have developed a microarray-based system for cell adhesion profiling of large panels of cell-adhesive proteins to increase the throughput of in vitro cell adhesion assays, which are currently primarily performed in multiwell plates. Miniaturizing cell adhesion assays to an array format required the development of protocols for the reproducible microspotting of extracellular matrix (ECM) protein solutions and for the handling of cell suspensions during the assay. We generated ECM protein microarrays with high reproducibility in microspot protein content using nitrocellulose-coated glass microslides, combined with piezoelectric microspotting of protein solutions. Protocols were developed that allowed us to use 5000 cells or fewer on an array of 4 x 4 mm consisting of 64 microspots. Using this microarray system, we identified differences of adhesive properties of three cell lines to 14 different ECM proteins. Furthermore, the sensitivity and accuracy of the assays were increased using microarrays with ranges of ECM protein amounts. This microarray system will be particularly useful for extensive comparative cell adhesion profiling studies when only low amounts of adhesive substrate and cells, such as stem cells or cells from biopsies, are available.     

Differentially Expressed RNA from Public Microarray Data Identifies Serum Protein Biomarkers for Cross-Organ Transplant Rejection and Other Conditions

Serum proteins are routinely used to diagnose diseases, but are hard to find due to low sensitivity in screening the serum proteome. Public repositories of microarray data, such as the Gene Expression Omnibus (GEO), contain RNA expression profiles for more than 16,000 biological conditions, covering more than 30% of United States mortality. We hypothesized that genes coding for serum- and urine-detectable proteins, and showing differential expression of RNA in disease-damaged tissues would make ideal diagnostic protein biomarkers for those diseases. We showed that predicted protein biomarkers are significantly enriched for known diagnostic protein biomarkers in 22 diseases, with enrichment significantly higher in diseases for which at least three datasets are available. We then used this strategy to search for new biomarkers indicating acute rejection (AR) across different types of transplanted solid organs. We integrated three biopsy-based microarray studies of AR from pediatric renal, adult renal and adult cardiac transplantation and identified 45 genes upregulated in all three. From this set, we chose 10 proteins for serum ELISA assays in 39 renal transplant patients, and discovered three that were significantly higher in AR. Interestingly, all three proteins were also significantly higher during AR in the 63 cardiac transplant recipients studied. Our best marker, serum PECAM1, identified renal AR with 89% sensitivity and 75% specificity, and also showed increased expression in AR by immunohistochemistry in renal, hepatic and cardiac transplant biopsies. Our results demonstrate that integrating gene expression microarray measurements from disease samples and even publicly-available data sets can be a powerful, fast, and cost-effective strategy for the discovery of new diagnostic serum protein biomarkers.     

Zeptosens' protein microarrays: a novel high performance microarray platform for low abundance protein analysis

Protein microarrays are considered an enabling technology, which will significantly expand the scope of current protein expression and protein interaction analysis. Current technologies, such as two-dimensional gel electrophoresis (2-DE) in combination with mass spectrometry, allowing the identification of biologically relevant proteins, have a high resolving power, but also considerable limitations. As was demonstrated by Gygi et al. (Proc. Nat. Acad. Sci. USA 2000,97, 9390-9395), most spots in 2-DE, observed from whole cell extracts, are from high abundance proteins, whereas low abundance proteins, such as signaling molecules or kinases, are only poorly represented. Protein microarrays are expected to significantly expedite the discovery of new markers and targets of pharmaceutical interest, and to have the potential for high-throughput applications. Key factors to reach this goal are: high read-out sensitivity for quantification also of low abundance proteins, functional analysis of proteins, short assay analysis times, ease of handling and the ability to integrate a variety of different targets and new assays. Zeptosens has developed a revolutionary new bioanalytical system based on the proprietary planar waveguide technology which allows us to perform multiplexed, quantitative biomolecular interaction analysis with highest sensitivity in a microarray format upon utilizing the specific advantages of the evanescent field fluorescence detection. The analytical system, comprising an ultrasensitive fluorescence reader and microarray chips with integrated microfluidics, enables the user to generate a multitude of high fidelity data in applications such as protein expression profiling or investigating protein-protein interactions. In this paper, the important factors for developing high performance protein microarray systems, especially for targeting low abundant messengers of relevant biological information, will be discussed and the performance of the system will be demonstrated in experimental examples.     

Experimental approach for assessing the outcome accuracy of antibody microarray experiments

An experimental strategy for quality control of antibody microarray analyses is proposed. The method utilizes proteins that are prepared for regular antibody microarray experiments. There is no need to use exogenous positive or negative reference markers and no need to determine the absolute concentration of each individual protein in the sample. Validation experiments support the basic principle of the proposed approach. This method can be a useful tool for assessing the outcome accuracy of microarray experiments.     

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.     

采用亲和层析结合制备凝胶电泳获得高纯度的重组抗原蛋白用于制备蛋白质芯片

为了得到制备抗原芯片所需的高纯度重组抗原蛋白,需要建立一套适合于多种重组抗原表达和纯化的技术路线．采用了亲和层析结合制备胶电泳的方法,对16种用于构建蛋白质芯片的食管癌相关抗原基因进行了克隆重组并在大肠杆菌中进行了表达．对高表达的重组蛋白首先制备包涵体,然后采用Ni-Sepharose亲和层析得到初步纯化的蛋白质,最后使用SDS-PAGE制备胶电泳作进一步纯化．经过透析复性后,用于制备蛋白质芯片．采用亲和层析纯化重组蛋白,得率为71% ,纯度约为70%;在SDS-PAGE制备胶进一步纯化后,得率为32%,纯度为95%,经过透析和复性后,最终得率为21%,纯度为95%．得到的重组蛋白RPS4在ELISA检测中可以和血清中识别RPS4 的自身抗体起反应,并且,采用精纯抗原制备的蛋白质芯片,在检测抗原与抗体这一对反应中也具有较高的敏感性和特异性,适合大规模血清抗体的检测．研究表明,采用亲和层析结合制备凝胶电泳纯化抗原蛋白,是一条简便快捷,适合需要量不大,但对纯度要求比较高的蛋白质芯片制备的技术路线．     

Profiling of alopecia areata autoantigens based on protein microarray technology

Protein biochips have a great potential in future parallel processing of complex samples as a research tool and in diagnostics. For the generation of protein biochips, highly automated technologies have been developed for cDNA expression library production, high throughput protein expression, large scale analysis of proteins, and protein microarray generation. Using this technology, we present here a strategy to identify potential autoantigens involved in the pathogenesis of alopecia areata, an often chronic disease leading to the rapid loss of scalp hair. Only little is known about the putative autoantigen(s) involved in this process. By combining protein microarray technology with the use of large cDNA expression libraries, we profiled the autoantibody repertoire of sera from alopecia areata patients against a human protein array consisting of 37,200 redundant, recombinant human proteins. The data sets obtained from incubations with patient sera were compared with control sera from clinically healthy persons and to background incubations with anti-human IgG antibodies. From these results, a smaller protein subset was generated and subjected to qualitative and quantitative validation on highly sensitive protein microarrays to identify novel alopecia areata-associated autoantigens. Eight autoantigens were identified by protein chip technology and were successfully confirmed by Western blot analysis. These autoantigens were arrayed on protein microarrays to generate a disease-associated protein chip. To confirm the specificity of the results obtained, sera from patients with psoriasis or hand and foot eczema as well as skin allergy were additionally examined on the disease-associated protein chip. By using alopecia areata as a model for an autoimmune disease, our investigations show that the protein microarray technology has potential for the identification and evaluation of autoantigens as well as in diagnosis such as to differentiate alopecia areata from other skin diseases.     

Protein profiling in human sera for identification of potential lung cancer biomarkers using antibody microarray

To identify potential biomarkers of lung cancer (LC), profiling of proteins in sera obtained from healthy and LC patients was determined using an antibody microarray. Based on our previous study on mRNA expression profiles between patients with LC and healthy persons, 19 proteins of interest were selected as targets for fabrication of an antibody microarray. Antibody to each protein and five nonspecific control antibodies were spotted onto a hydrogel‐coated glass slide and used for profiling of proteins in sera of LC patients in a two‐color fluorescence assay. Forty‐eight human sera samples were analyzed, and expression profiling of proteins were represented by the internally normalized ratio method. Six proteins were distinctly down‐regulated in sera of LC patients; this observation was validated by Wilcoxon test, false discovery rate, and Western blotting. Blind test of other 32 human sera using the antibody microarray followed by hierarchical clustering analysis revealed an approximate sensitivity of 88%, specificity of 80%, and an accuracy of 84%, respectively, in classifying the sera, which supports the potential of the six identified proteins as biomarkers for the prognosis of lung cancer.     

Use of protein-acrylamide copolymer hydrogels for measuring protein concentration and activity

We report the development and characterization of a polyacrylamide-based protein immobilization strategy for surface-bound protein assays, including concentration detection, binding affinity, and enzyme kinetics. Glutathione S-transferase (GST) fusion proteins have been labeled with an acrylic moiety and attached to acrylic-functionalized glass surfaces through copolymerization with acrylic monomer. The specific attachment of GST-green fluorescent protein (GFP) fusion protein was more than sevenfold greater than the nonspecific attachment of nonacrylic-labeled GST-GFP; 0.32 ng/mm(2) of surface-attached GST-GFP was detectable by direct measurement of GFP fluorescence and this lower detection limit was reduced to 0.080 ng/mm(2) using indirect antibody-based detection. The polyacrylamide-based surface attachment strategy was also used to measure the kinetics of substrate phosphorylation by the kinase c-Src. Michaelis-Menten kinetic constants for the reaction occurring in solution were K(m) = 2.7 +/- 1.0 microM and V(max) = 8.1 +/- 3.1 (arbitrary units). Kinetic values for the reaction utilizing surface-immobilized substrate were K(m) = 0.36 +/- 0.033 microM and V(max) = 9.7 +/- 0.63 and were found to be independent of the acrylamide concentration within the copolymer. Such a surface attachment strategy should be applicable to the proteomics field and addresses denaturation and dehydration problems associated with protein microarray development.     

The use of lectin microarray for assessing glycosylation of therapeutic proteins

Glycans or carbohydrates attached to therapeutic glycoproteins can directly affect product quality, safety and efficacy, and therefore must be adequately analyzed and controlled throughout product life cycles. However, the complexity of protein glycosylation poses a daunting analytical challenge. In this study, we evaluated the utility of a lectin microarray for assessing protein glycans. Using commercial lectin chips, which contain 45 lectins toward distinct glycan structures, we were able to determine the lectin binding patterns of a panel of 15 therapeutic proteins, including 8 monoclonal antibodies. Lectin binding signals were analyzed to generate glycan profiles that were generally consistent with the known glycan patterns for these glycoproteins. In particular, the lectin-based microarray was found to be highly sensitive to variations in the terminal carbohydrate structures such as galactose versus sialic acid epitopes. These data suggest that lectin microarray could be used for screening glycan patterns of therapeutic glycoproteins.     

Identification of new autoantigens for primary biliary cirrhosis using human proteome microarrays

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease of unknown etiology and is considered to be an autoimmune disease. Autoantibodies are important tools for accurate diagnosis of PBC. Here, we employed serum profiling analysis using a human proteome microarray composed of about 17,000 full-length unique proteins and identified 23 proteins that correlated with PBC. To validate these results, we fabricated a PBC-focused microarray with 21 of these newly identified candidates and nine additional known PBC antigens. By screening the PBC microarrays with additional cohorts of 191 PBC patients and 321 controls (43 autoimmune hepatitis, 55 hepatitis B virus, 31 hepatitis C virus, 48 rheumatoid arthritis, 45 systematic lupus erythematosus, 49 systemic sclerosis, and 50 healthy), six proteins were confirmed as novel PBC autoantigens with high sensitivities and specificities, including hexokinase-1 (isoforms I and II), Kelch-like protein 7, Kelch-like protein 12, zinc finger and BTB domain-containing protein 2, and eukaryotic translation initiation factor 2C, subunit 1. To facilitate clinical diagnosis, we developed ELISA for Kelch-like protein 12 and zinc finger and BTB domain-containing protein 2 and tested large cohorts (297 PBC and 637 control sera) to confirm the sensitivities and specificities observed in the microarray-based assays. In conclusion, our research showed that a strategy using high content protein microarray combined with a smaller but more focused protein microarray can effectively identify and validate novel PBC-specific autoantigens and has the capacity to be translated to clinical diagnosis by means of an ELISA-based method.