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Results for “Protein Array Analysis”

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At least 19 recordsLinked to original sources

PubMed2005

Patterns of protein expression in infectious meningitis: a cerebrospinal fluid protein array analysis.

Seventy-nine cytokines, chemokines, and growth factors were measured by protein array analysis in the cerebrospinal fluid of patients with meningitis and controls. Several factors were found to be regulated, which have not been studied in the CNS before, e.g., macrophage inflammatory protein-1delta (CCL15) and neutrophil-activating peptide-2 (CXCL7). In pneumococcal meningitis, other new observations were an increase of macrophage migration inhibitory factor, monocyte chemoattractant protein-2 (CCL8), pulmonary and activation-regulated chemokine (CCL18), and macrophage inflammatory protein-3alpha (CCL20), and a sustained upregulation of several growth factors. In viral meningitis, new findings were an elevation of CCL8, thrombopoietin, and vascular endothelial growth factor.

Chemokines↗

PubMed2006

Donor-reactive cytokine production after HLA-identical living related kidney transplantation: a protein-array analysis.

In the present pilot study, we investigated which proteins are produced after donor stimulation of peripheral blood mononuclear cells from recipients of HLA-identical living related kidney transplant. We used a protein-array analysis to determine cytokines, chemokines, and growth factors in supernatant from donor-stimulated mixed lymphocyte reaction cultures. Autologous cultures were considered to be negative controls. In 38 out of 42 proteins (90%), the donor response was higher compared with the autologous response. Therefore, we concluded that even after HLA-identical living related kidney transplantation we could measure a donor-reactive response, which we assumed was directed toward minor histocompatibility or non-HLA antigens.

Chemokines↗

PubMed2006

Surface plasmon resonance imaging protein arrays for analysis of triple protein interactions of HPV, E6, E6AP, and p53.

We have developed a surface plasmon resonance (SPR)-based protein microarray to study protein-protein interactions in a high-throughput mode. As a model system, triple protein interactions have been explored with human papillomaviral E6 protein, tumor suppressor p53, and ubiquitin ligase E6AP. Human papillomavirus (HPV) is known to be a causative agent of cervical cancer. Upon infection, the viral E6 protein forms a heterotrimeric protein complex with p53 and E6AP. The formation of the complex eventually results in the degradation of p53. In the present study, a GST-fused E6AP protein was layered onto a glutathione (GSH)-modified gold chip surface. The specific binding of GST-E6AP protein onto the gold chip surface was facilitated through the affinity of GST to its specific ligand GSH. The interacting proteins (E6 and/or p53) were then spotted. Detection of the interaction was performed using a SPR imaging (SPRI) technique. The resulting SPRI intensity data showed that the protein-protein interactions of E6AP, E6, and p53 were detected in a concentration-dependent manner, suggesting that the SPRI-based microarray system can be an effective tool to study protein-protein interactions where multiple proteins are involved.

Humans↗

PubMed2005

Sensitivity of ex situ and in situ spectral surface plasmon resonance sensors in the analysis of protein arrays.

We have investigated the sensitivity of ex situ (analysis under air condition) and in situ (analysis under liquid condition) spectral SPR sensors, which were self-constructed with fiber optic spectrometers. The sensitivity of SPR sensors was analyzed in the wavelength range of 550-780 nm by the interactions of streptavidin and biotinylated IgG, and the sensitivity was dependent on the wavelength of measurements. The sensitivity of an ex situ SPR sensor operated at the long wavelength range from 712 nm was approximately 2.6 times higher than that at the short wavelength range from 571 nm. In addition, the sensitivity of an ex situ spectral SPR sensor was about twice as high as that of an in situ spectral SPR sensor for the same resonance wavelength range. This was interpreted in that the difference in sensitivity between two SPR sensors was significantly caused by the evanescent field intensity at the metal/dielectric interface. Thus, it was suggested that ex situ spectral SPR sensors operated at the long wavelength range are sensitive biosensors for the high-throughput analysis of protein interactions on protein arrays.

Biosensing Techniques↗

PubMed2006

Cluster analysis of protein array results via similarity of Gene Ontology annotation.

BACKGROUND: With the advent of high-throughput proteomic experiments such as arrays of purified proteins comes the need to analyse sets of proteins as an ensemble, as opposed to the traditional one-protein-at-a-time approach. Although there are several publicly available tools that facilitate the analysis of protein sets, they do not display integrated results in an easily-interpreted image or do not allow the user to specify the proteins to be analysed. RESULTS: We developed a novel computational approach to analyse the annotation of sets of molecules. As proof of principle, we analysed two sets of proteins identified in published protein array screens. The distance between any two proteins was measured as the graph similarity between their Gene Ontology (GO) annotations. These distances were then clustered to highlight subsets of proteins sharing related GO annotation. In the first set of proteins found to bind small molecule inhibitors of rapamycin, we identified three subsets containing four or five proteins each that may help to elucidate how rapamycin affects cell growth whereas the original authors chose only one novel protein from the array results for further study. In a set of phosphoinositide-binding proteins, we identified subsets of proteins associated with different intracellular structures that were not highlighted by the analysis performed in the original publication. CONCLUSION: By determining the distances between annotations, our methodology reveals trends and enrichment of proteins of particular functions within high-throughput datasets at a higher sensitivity than perusal of end-point annotations. In an era of increasingly complex datasets, such tools will help in the formulation of new, testable hypotheses from high-throughput experimental data.

Algorithms↗

PubMed2005

Proteomic applications of surface plasmon resonance biosensors: analysis of protein arrays.

Proteomics is one of the most important issues in the post-genomic area, because it can greatly contribute to identifying protein biomarkers for disease diagnosis and drug screening. Protein array is a key technology for proteome researches and has been analyzed by various methods including fluorescence, mass spectrometry, atomic force microscopy and surface plasmon resonance (SPR). SPR biosensor is a promising technology in proteomics, since it has various advantages including real-time measurement of biomolecular interactions without labeling and the simple optical system for the device. SPR biosensors have a strong potential for analyzing proteomes by SPR imaging and SPR spectroscopic imaging, even though the challenge is to produce proteins on a proteomic scale.

Animals↗

PubMed2004

Analysis of protein interactions on protein arrays by a wavelength interrogation-based surface plasmon resonance biosensor.

We have investigated whether surface plasmon resonance (SPR) sensors based on the wavelength interrogation are able to analyze protein interactions on protein arrays. The spectral SPR sensor was self-constructed and its detection limit, expressed as the minimal refractive index variation, was calculated to be 6.6x10(-5) with the signal fluctuation of 1.0x10(-5). The protein array surface was modified by a mixed thiol monolayer to immobilize proteins. Protein arrays were analyzed by the line-scanning mode of the SPR sensor, which scanned every 100 microm along the central line of array spots and the scanned results were presented by color spectra from blue to red. Glutathione S-transferase (GST)-rac1 caused a concentration-dependent increase of SPR wavelength shift on protein arrays. The surface structure of the protein arrays was analyzed by atomic force microscopy. Specific interactions of antigens with antibodies were analyzed on the protein arrays by using three antibodies and eight proteins. These results suggest that the wavelength interrogation-based SPR sensor can be used as the biosensor for the high-throughput analysis of protein interactions on protein arrays.

Antibodies↗

PubMed2006

Sensitivity enhancement of spectral surface plasmon resonance biosensors for the analysis of protein arrays.

A novel method for sensitivity enhancement of spectral surface plasmon resonance (SPR) biosensors was presented by reducing the refractive index of the sensing prism in the analysis of protein arrays. Sensitivity of spectral SPR biosensors with two different prisms (BK-7, fused silica) was analyzed by net shifts of resonance wavelength for specific interactions of GST-GTPase binding domain of p21-activated kinase-1 and anti-GST on a mixed thiol surface. Sensitivity was modulated by the refractive index of the sensing prism of the spectral SPR biosensors with the same incidence angle. The sensitivity of a spectral SPR biosensor with a fused silica prism was 1.6 times higher than that with a BK-7 prism at the same incidence angle of 46.2 degrees. This result was interpreted by increment of the penetration depth correlated with evanescent field intensity at the metal/dielectric interface. Therefore, it is suggested that sensitivity enhancement is readily achieved by reducing the refractive index of the sensing prism of spectral SPR biosensors to be operated at long wavelength ranges for the analysis of protein arrays.

Biophysics↗

PubMed2005

A novel peptide microarray for protein detection and analysis utilizing a dry peptide array system.

A novel dry peptide microarray system has been constructed that affords a practical solution for protein detection and analysis. This system is an array preparation and assay procedure under dry conditions that uses designed peptides as non-immobilized capture agents for the detection of proteins. The system has several advantages that include its portability and ease-of-use, as well as the fact that vaporization of sample solutions need not be considered. In this study, various proteins have been characterized with an alpha-helical peptide mini-library. When proteins were added to the peptide library array, the fluorescent peptides showed different fluorescent intensities depending on their sequences. The patterns of these responses could be regarded as 'protein fingerprints' (PFPs), which are sufficient to establish the identities of the target proteins. Furthermore, statistical analysis of the resulting PFPs was performed using cluster analysis. The PFPs of the proteins were clustered successfully depending on their families and binding properties. Additionally, the target protein was characterized using a nanolitre system and could be detected down to 1.2 fmol. These studies imply that the dry peptide array system is a promising tool for detecting and analyzing target proteins. The dry peptide array will play a role in development of high-throughput protein-detecting nano/micro arrays for proteomics and ligand screening studies.

Amino Acid Sequence↗

PubMed2002

Frozen protein arrays: a new method for arraying and detecting recombinant and native tissue proteins.

DNA microarrays are powerful tools for high throughput analysis of gene expression; however, they do not measure protein expression. Current methods for producing protein arrays require sophisticated equipment or extensive protein modification. We developed a low overhead, customizable assay platform called frozen protein arrays that can detect native proteins in protein lysates. Frozen protein arrays were formed from a block of frozen histologic embedding compound containing an array of wells. The wells were filled with samples, which freeze and bond to the block. Cryosections were cut and transferred to nitrocellulose-coated slides. The reproducibility, linearity, and sensitivity was confirmed using frozen protein arrays filled with prostate specific antigen. Frozen protein arrays could detect native tissue proteins. The alpha1 subunit of NaK-ATPase was detected in rat kidneys with a coefficient of variation of 4.3-6.6%. Frozen protein array analysis indicated that the protein abundance decreased by 48.7% following renal ischemia, similar to the 40% decrease by Western blotting. We conclude that frozen protein arrays are a low cost, moderate size platform for arraying samples including protein lysates. Production of many identical frozen protein arrays is easy, inexpensive, and requires only small sample volumes. The method is gentle on proteins as they remain frozen during production.

Animals↗

PubMed2005

Analysis of protein interactions on protein arrays by a novel spectral surface plasmon resonance imaging.

We presented a novel surface plasmon resonance (SPR) imaging method for analysis of protein arrays based on a wavelength interrogation-based SPR biosensor. The spectral imaging was performed by the combination of position control and resonance wavelengths calculated from SPR reflectivity spectra. The imaging method was evaluated by analyzing interactions of glutathione S-transferase-fusion proteins with their antibodies. Antigen-antibody interactions were successfully analyzed on glutathione S-transferase-fusion protein arrays by using the spectral imaging method, and the results were confirmed by a parallel analysis using a previously used spectral SPR biosensor based on wavelength interrogation. Specific binding of anti-Rac1 and anti-RhoA to Rac1 and RhoA on the protein arrays was qualitatively and quantitatively analyzed by the spectral SPR imaging. Thus, it was suggested that the novel spectral SPR imaging was a useful tool for the high-throughput analysis of protein-protein interactions on protein arrays.

Antigen-Antibody Complex↗

PubMed2002

Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions.

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.

3-Phosphoinositide-Dependent Protein Kinases↗

PubMed2004

A versatile polypeptide platform for integrated recognition and reporting: affinity arrays for protein-ligand interaction analysis.

A molecular platform for protein detection and quantification is reported in which recognition has been integrated with direct monitoring of target-protein binding. The platform is based on a versatile 42-residue helix-loop-helix polypeptide that dimerizes to form four-helix bundles and allows site-selective modification with recognition and reporter elements on the side chains of individually addressable lysine residues. The well-characterized interaction between the model target-protein carbonic anhydrase and its inhibitor benzenesulfonamide was used for a proof-of-concept demonstration. An affinity array was designed where benzenesulfonamide derivatives with aliphatic or oligoglycine spacers and a fluorescent dansyl reporter group were introduced into the scaffold. The affinities of the array members for human carbonic anhydrase II (HCAII) were determined by titration with the target protein and were found to be highly affected by the properties of the spacers (dissociation constant Kd=0.02-3 microM). The affinity of HCAII for acetazolamide (Kd=4 nM) was determined in a competition experiment with one of the benzenesulfonamide array members to address the possibility of screening substance libraries for new target-protein binders. Also, successful affinity discrimination between different carbonic anhydrase isozymes highlighted the possibility of performing future isoform-expression profiling. Our platform is predicted to become a flexible tool for a variety of biosensor and protein-microarray applications within biochemistry, diagnostics and pharmaceutical chemistry.

Acetazolamide↗

PubMed2005

Mining ChIP-chip data for transcription factor and cofactor binding sites.

MOTIVATION: Identification of single motifs and motif pairs that can be used to predict transcription factor localization in ChIP-chip data, and gene expression in tissue-specific microarray data. RESULTS: We describe methodology to identify de novo individual and interacting pairs of binding site motifs from ChIP-chip data, using an algorithm that integrates localization data directly into the motif discovery process. We combine matrix-enumeration based motif discovery with multivariate regression to evaluate candidate motifs and identify motif interactions. When applied to the HNF localization data in liver and pancreatic islets, our methods produce motifs that are either novel or improved known motifs. All motif pairs identified to predict localization are further evaluated according to how well they predict expression in liver and islets and according to how conserved are the relative positions of their occurrences. We find that interaction models of HNF1 and CDP motifs provide excellent prediction of both HNF1 localization and gene expression in liver. Our results demonstrate that ChIP-chip data can be used to identify interacting binding site motifs. AVAILABILITY: Motif discovery programs and analysis tools are available on request from the authors.

Algorithms↗

PubMed2006

Prediction of renal allograft rejection by urinary protein analysis using ProteinChip Arrays (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry).

OBJECTIVES: To develop a noninvasive method for the detection of renal transplant rejection using ProteinChip Arrays (surface-enhanced laser desorption/ionization time-of-flight mass spectrometry). METHODS: A total of 23 urine samples were collected from 13 patients showing biopsy-proven renal allograft rejection and from 10 patients without histologic signs of rejection. All 23 patients had clinical symptoms and signs of acute allograft rejection and underwent renal biopsy. Samples were centrifuged, and supernatants were directly spotted onto the ProteinChip arrays with different chromatographic surfaces. The obtained spectra in a range from 2 to 200 kDa were subjected to bioinformatic analysis using the method of Fuzzy c-means, followed by the establishment of rule bases and evaluation using the relevance index according to Kiendl. RESULTS: Several protein peaks were identified allowing differentiation between rejection and no rejection. Using two different ProteinChip surfaces, we found two biomarkers at 25.71 kDa and 28.13 kDa that gave a diagnostic sensitivity of 90% and 93% and a specificity of 80% (SAX2) and 85% (CM10), respectively. CONCLUSIONS: Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry appears to be a promising new diagnostic tool for distinguishing renal transplant patients with no rejection from those with acute rejection.

Graft Rejection↗

PubMed2005

Experimental design and analysis of antibody microarrays: applying methods from cDNA arrays.

Protein expression microarrays, also called antibody arrays, represent a new technology that allows the expression level of proteins to be assessed directly. As is also the case with gene expression microarrays, it is hoped that protein expression microarrays will aid in biomarker discovery, predicting disease outcomes and response to treatments, and detecting molecular mechanisms and/or pathways associated with a particular disease state. However, accurately achieving these aims is dependent upon suitable experimental designs, normalization procedures that eliminate systematic bias, and appropriate statistical analyses to assess differential expression or expose expression patterns. In the last five years, a large amount of research has been devoted to two-color cDNA arrays to improve experimental design, normalization and statistical analyses to assess differential expression and classification. These methods are directly applicable to two-color antibody arrays. The objective of this article is to discuss statistical methods that have been developed for cDNA arrays and describe how the methods can be directly applied to antibody arrays.

Antibodies↗

PubMed2005

Analysis of protein interaction and function with a 3-dimensional MALDI-MS protein array.

Protein profiling and characterization of protein interactions in biological samples ultimately require indicator-free methods of signal detection, which likewise offer an opportunity to distinguish specific interactions from nonspecific protein binding. Here we describe a new 3-dimensional protein microchip for detecting biomolecular interactions with matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS); the microchip comprises a high-density array of methacrylate polymer elements containing immobilized proteins as capture molecules and directly interfaces with a commercially available mass spectrometer. We demonstrated the performance of the chip in three types of experiments by detecting antibody-antigen interactions, enzymatic activity, and enzyme-inhibitor interactions. MALDI-MS biochip-based tumor necrosisfactor alpha (TNF-alpha) immunoassays demonstrated the feasibility of detecting antigens in complex biological samples by identifying molecular masses of bound proteins even at high nonspecific protein binding. By detecting model interactions of trypsin with trypsin inhibitors, we showed that the protein binding capacity of methacrylate polymer elements and the sensitivity of MALDI-MS detection of proteins bound to these elements surpassed that of other 2- and 3-dimensional substrates tested Immobilized trypsin retained functional (enzymatic) activity within the protein microchip and the specificity of macromolecular interactions even in complex biological samples. We believe that the underlying technology should therefore be extensible to whole-proteome protein expression profiling and interaction mapping.

Equipment Design↗

PubMed2003

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.

Binding Sites↗