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Results for “Protein Array Analysis”

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At least 1,009 records · Page 56Linked to original sources

PubMed2006

A red-fluorescent substrate microarray for lipase fingerprinting.

A lipase substrate microarray was obtained by printing aliphatic C2-C12 monoesters of (5R)- and (5S)-3-(5,6-dihydroxyhexyloxy)benzaldehyde by reductive alkylation on amine-functionalized glass slides coated with bovine serum albumin and a short PEG linker. The microarray features 12 substrates and their 66 possible binary mixtures spotted in a 9 x 36 spot array. Lipase reactions are detected by chemoselective NaIO(4)-oxidation of the 1,2-diol hydrolysis product to form an aldehyde, which is then tagged with the red-fluorescent dye rhodamine B sulfohydrazide . Specific fingerprints are produced by active enzymes. These experiments provide the first example of lipase fingerprinting using microarrays.

Cluster Analysis↗

PubMed2005

Design and simulation of active biochip system.

To solve the problems existing in passive biochip systems, we designed a novel active biochip system. This system introduces negative pressure and controlling devices to adjust the antigen-antibody reaction on the nitrocellulose membrane. Computational simulation demonstrated that this system is a rapid, stable, robust and practical system that may enhance the efficiency of antigen-antibody reactions and improve the repeatability and accuracy of biochip analysis.

Antigen-Antibody Complex↗

PubMed2006

SELDI-TOF-MS of saliva: methodology and pre-treatment effects.

Interest in saliva as a diagnostic fluid for monitoring general health and for early diagnosis of disease has increased in the last few years. In particular, efforts have focused on the generation of protein maps of saliva using advanced proteomics technology. Surface-enhanced laser-desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) is a novel high throughput and extremely sensitive proteomic approach that allows protein expression profiling of large sets of complex biological specimens. In this study, large scale profiling of salivary proteins and peptides, ranging from 2 to 100kDa was demonstrated using SELDI-TOF-MS. Various methodological aspects and pre-analytical variables were analysed with respect to their effects on saliva SELDI-TOF-MS profiling. Results show that chip surface type and sample type (unstimulated versus stimulated) critically affect the amount and composition of detected salivary proteins. Factors that influenced normal saliva protein profiling were matrix composition, sample dilution and binding buffer properties. Delayed processing time experiments show certain new peptides evolving 3h post-saliva donation, and quantitative analyses indicate relative intensity of other proteins and peptides changing with time. The addition of protease inhibitors partly counteracted the destabilization of certain protein/peptide mass spectra over time suggesting that some proteins in saliva are subject to digestion by intrinsic salivary proteases. SELDI-TOF-MS profiles also changed by varying storage time and storage temperature whereas centrifugation speed and freeze-thaw cycles had minimal impact. In conclusion, SELDI-TOF-MS offers a high throughput platform for saliva protein and peptide profiling, however, (pre-)analytical conditions must be taken into account for valid interpretation of the acquired data.

Biomarkers↗

PubMed2003

Human SPF45, a splicing factor, has limited expression in normal tissues, is overexpressed in many tumors, and can confer a multidrug-resistant phenotype to cells.

Our effort to identify novel drug-resistant genes in cyclophosphamide-resistant EMT6 mouse mammary tumors led us to the identification of SPF45. Simultaneously, other groups identified SPF45 as a component of the spliceosome that is involved in alternative splicing. We isolated the human homologue and examined the normal human tissue expression, tumor expression, and the phenotype caused by overexpression of human SPF45. Our analyses revealed that SPF45 is expressed in many, but not all, normal tissues tested with predominant expression in normal ductal epithelial cells of the breast, liver, pancreas, and prostate. Our analyses using tissue microarrays and sausages of tumors indicated that SPF45 is highly expressed in numerous carcinomas including bladder, breast, colon, lung, ovarian, pancreatic, and prostate. Interestingly, this study revealed that overexpression of SPF45 in HeLa, a cervical carcinoma cell line, resulted in drug resistance to doxorubicin and vincristine, two chemotherapeutic drugs commonly used in cancer. To our knowledge, this is the first study showing tumor overexpression of an alternate splicing factor resulting in drug resistance.

Blotting, Western↗

PubMed2004

Development and evaluation of a protein microarray chip for diagnosis of hepatitis C virus.

A protein chip diagnostic kit was developed for the diagnosis of hepatitis C virus (HCV) based on the protein chip technique and the immuno-concentration method. This kit was designed for low-density protein chips and also for the availability of multiple sample screening. Applicability of the chip was evaluated using 96 blood specimens and the results were compared to results of an anti-HCV enzyme immunoassay (EIA) test. With further development, the technology associated with the development of this chip could be applied to the simultaneous detection of multiple protein-protein, protein-ligand interactions.

Enzyme-Linked Immunosorbent Assay↗

PubMed2006

Protein biomarker discovery and validation: the long and uncertain path to clinical utility.

Better biomarkers are urgently needed to improve diagnosis, guide molecularly targeted therapy and monitor activity and therapeutic response across a wide spectrum of disease. Proteomics methods based on mass spectrometry hold special promise for the discovery of novel biomarkers that might form the foundation for new clinical blood tests, but to date their contribution to the diagnostic armamentarium has been disappointing. This is due in part to the lack of a coherent pipeline connecting marker discovery with well-established methods for validation. Advances in methods and technology now enable construction of a comprehensive biomarker pipeline from six essential process components: candidate discovery, qualification, verification, research assay optimization, biomarker validation and commercialization. Better understanding of the overall process of biomarker discovery and validation and of the challenges and strategies inherent in each phase should improve experimental study design, in turn increasing the efficiency of biomarker development and facilitating the delivery and deployment of novel clinical tests.

Animals↗

PubMed2005

Vaccinia virus H3L envelope protein is a major target of neutralizing antibodies in humans and elicits protection against lethal challenge in mice.

The smallpox vaccine is the prototypic vaccine, yet the viral targets critical for vaccine-mediated protection remain unclear in humans. We have produced protein microarrays of a near-complete vaccinia proteome and used them to determine the major antigen specificities of the human humoral immune response to the smallpox vaccine (Dryvax). H3L, an intracellular mature virion envelope protein, was consistently recognized by high-titer antibodies in the majority of human donors, particularly after secondary immunization. We then focused on examining H3L as a valuable human antibody target. Purified human anti-H3L antibodies exhibited substantial vaccinia virus-neutralizing activity in vitro (50% plaque reduction neutralization test [PRNT50] = 44 microg/ml). Mice also make an immunodominant antibody response to H3L after vaccination with vaccinia virus, as determined by vaccinia virus protein microarray. Mice were immunized with recombinant H3L protein to examine H3L-specific antibody responses in greater detail. H3L-immunized mice developed high-titer vaccinia virus-neutralizing antibodies (mean PRNT50 = 1:3,760). Importantly, H3L-immunized mice were subsequently protected against lethal intranasal challenges with 1 or 5 50% lethal doses (LD50) of pathogenic vaccinia virus strain WR, demonstrating the in vivo value of an anti-H3L response. To formally demonstrate that neutralizing anti-H3L antibodies are protective in vivo, we performed anti-H3L serum passive-transfer experiments. Mice receiving H3L-neutralizing antiserum were protected from a lethal challenge with 3 LD50 of vaccinia virus strain WR (5/10 versus 0/10; P < 0.02). Together, these data show that H3L is a major target of the human anti-poxvirus antibody response and is likely to be a key contributor to protection against poxvirus infection and disease.

Administration, Intranasal↗

PubMed2004

Functional analysis of the alpha-defensin disulfide array in mouse cryptdin-4.

The alpha-defensin antimicrobial peptide family is defined by a unique tridisulfide array. To test whether this invariant structural feature determines alpha-defensin bactericidal activity, mouse cryptdin-4 (Crp4) tertiary structure was disrupted by pairs of site-directed Ala for Cys substitutions. In a series of Crp4 disulfide variants whose cysteine connectivities were confirmed using NMR spectroscopy and mass spectrometry, mutagenesis did not induce loss of function. To the contrary, the in vitro bactericidal activities of several Crp4 disulfide variants were equivalent to or greater than those of native Crp4. Mouse Paneth cell alpha-defensins require the proteolytic activation of precursors by matrix metalloproteinase-7 (MMP-7), prompting an analysis of the relative sensitivities of native and mutant Crp4 and pro-Crp4 molecules to degradation by MMP-7. Although native Crp4 and the alpha-defensin moiety of proCrp4 resisted proteolysis completely, all disulfide variants were degraded extensively by MMP-7. Crp4 bactericidal activity was eliminated by MMP-7 cleavage. Thus, rather than determining alpha-defensin bactericidal activity, the Crp4 disulfide arrangement confers essential protection from degradation by this critical activating proteinase.

Amino Acid Sequence↗

PubMed2002

The predicted candidates of Arabidopsis plastid inner envelope membrane proteins and their expression profiles.

Plastid envelope proteins from the Arabidopsis nuclear genome were predicted using computational methods. Selection criteria were: first, to find proteins with NH(2)-terminal plastid-targeting peptides from all annotated open reading frames from Arabidopsis; second, to search for proteins with membrane-spanning domains among the predicted plastidial-targeted proteins; and third, to subtract known thylakoid membrane proteins. Five hundred forty-one proteins were selected as potential candidates of the Arabidopsis plastid inner envelope membrane proteins (AtPEM candidates). Only 34% (183) of the AtPEM candidates could be assigned to putative functions based on sequence similarity to proteins of known function (compared with the 69% function assignment of the total predicted proteins in the genome). Of the 183 candidates with assigned functions, 40% were classified in the category of "transport facilitation," indicating that this collection is highly enriched in membrane transporters. Information on the predicted proteins, tissue expression data from expressed sequence tags and microarrays, and publicly available T-DNA insertion lines were collected. The data set complements proteomic-based efforts in the increased detection of integral membrane proteins, low-abundance proteins, or those not expressed in tissues selected for proteomic analysis. Digital northern analysis of expressed sequence tags suggested that the transcript levels of most AtPEM candidates were relatively constant among different tissues in contrast to stroma and the thylakoid proteins. However, both digital northern and microarray analyses identified a number of AtPEM candidates with tissue-specific expression patterns.

Arabidopsis↗

PubMed2006

Protein expression pattern in experimental pneumococcal meningitis.

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.

Animals↗

PubMed2005

A software suite for the generation and comparison of peptide arrays from sets of data collected by liquid chromatography-mass spectrometry.

There is an increasing interest in the quantitative proteomic measurement of the protein contents of substantially similar biological samples, e.g. for the analysis of cellular response to perturbations over time or for the discovery of protein biomarkers from clinical samples. Technical limitations of current proteomic platforms such as limited reproducibility and low throughput make this a challenging task. A new LC-MS-based platform is able to generate complex peptide patterns from the analysis of proteolyzed protein samples at high throughput and represents a promising approach for quantitative proteomics. A crucial component of the LC-MS approach is the accurate evaluation of the abundance of detected peptides over many samples and the identification of peptide features that can stratify samples with respect to their genetic, physiological, or environmental origins. We present here a new software suite, SpecArray, that generates a peptide versus sample array from a set of LC-MS data. A peptide array stores the relative abundance of thousands of peptide features in many samples and is in a format identical to that of a gene expression microarray. A peptide array can be subjected to an unsupervised clustering analysis to stratify samples or to a discriminant analysis to identify discriminatory peptide features. We applied the SpecArray to analyze two sets of LC-MS data: one was from four repeat LC-MS analyses of the same glycopeptide sample, and another was from LC-MS analysis of serum samples of five male and five female mice. We demonstrate through these two study cases that the SpecArray software suite can serve as an effective software platform in the LC-MS approach for quantitative proteomics.

Amino Acid Sequence↗

PubMed2005

Validation and quality control of protein microarray-based analytical methods.

The microarray has emerged as an important format for simultaneous analysis of tens of thousands of substances present in a sample. Successful adaptation of microarray assays to clinical diagnostics will require particular attention to issues of quality control and quality assurance. Results of an assay can be compromised by a number of preanalytical factors including the quality of the reagents (e.g., the microarray and the detection reagents) and the integrity of the sample. Similarly, numerous factors in the analytical phase of a microarray assay, including changes in the reaction conditions and calibration, can contribute to inaccuracy and imprecision. Furthermore, a microarray combines many reagents or samples in a single device and therefore presents additional issues not usually encountered in discrete testing of a single analyte in a single sample. Various strategies (e.g., replicate analysis, array orientation control features, on-array controls, normalization) have been implemented to control and assess analytical factors that might compromise data generated from a microarray. The current range of measures taken to ensure the analytical accuracy and quality of data generated from protein microarrays is reviewed in the context of the lessons learned from DNA microarrays. The special considerations for protein microarrays as they transition from research into routine clinical analysis and the resulting quality control of clinical test results generated using such devices are discussed.

Computational Biology↗

PubMed2004

Multiplexed sandwich assays in microarray format.

Antibody arrays have evolved into powerful tools for quantifying proteins and qualifying their state of activation in complex biological samples. This level of analysis holds tremendous promise as part of a diagnostic or prognostic platform. In particular, multiplex sandwich ELISA assays performed with microarrays in the wells of multi-well plates enable high-throughput analysis of multiple samples with multivariate readouts. Here, we compare and review recent advances in antibody microarray technology and describe its promises towards the systematic analysis of complex biological samples, ranging from profiling patient sera to studying intracellular signaling.

Animals↗

PubMed2005

Protein arrays for studying blood cells and their secreted products.

Protein microarrays have been developed and partially validated for studying blood cells, which play a role in many human diseases. Arrays of capture antibodies are commercially available for analyzing cytokines and intracellular signaling proteins. Several academic laboratories have developed antigen microarrays for characterizing autoimmune and allergic diseases, with a goal toward using such arrays to profile antibodies found in blood or other biological fluids. Arrays composed of major histocompatibility complex tetramers have been constructed and validated for analysis of immune responses in mice, paving the way toward studying antigen-specific T-lymphocyte responses. Finally, reverse-phase protein lysate microarray technology, first developed for analyzing cancer cells from tissue sections, has now been demonstrated for studying living cells, including knockout cells, cells treated with drugs such as kinase inhibitors, and rare populations of lymphocytes such as regulatory T cells. The goal of this review is to focus on advances in and future uses of arrays of proteins that can be printed on glass microscope slides using traditional microarray robots that are commonly found at academic medical centers. Dissemination of protein array technology will occur in the next decade and will markedly change how immunology research, particularly in the fields of autoimmunity and inflammation, is conducted.

Animals↗

PubMed2003

Comprehensive proteomic profiling identifies serum proteomic signatures for detection of hepatocellular carcinoma and its subtypes.

BACKGROUND: Detection of hepatocellular carcinoma (HCC) in patients with chronic liver disease (CLD) is difficult. We investigated the use of comprehensive proteomic profiling of sera to differentiate HCC from CLD. METHODS: Proteomes in sera from 20 CLD patients with alpha-fetoprotein (AFP) <500 microg/L (control group) and 38 HCC patients (disease group) were profiled by anion-exchange fractionation (first dimension), two types (IMAC3 copper and WCX2) of ProteinChip Arrays (second dimension), and time-of-flight mass spectrometry (third dimension). Bioinformatic tests were used to identify tumor-specific proteomic features and to estimate the values of the tumor-specific proteomic features in the diagnosis of HCC. Cross-validation was performed, and we also validated the models with pooled sera from the control and disease groups, serum from a CLD patient with AFP >500 microg/L, and postoperative sera from two HCC patients. RESULTS: Among 2384 common serum proteomic features, 250 were significantly different between the HCC and CLD cases. Two-way hierarchical clustering differentiated HCC and CLD cases. Most HCC cases with advanced disease were clustered together and formed two subgroups that contained significantly more cases with lymph node invasion or distant metastasis. For differentiation of HCC and CLD by an artificial network (ANN), the area under the ROC curve was 0.91 (95% confidence interval, 0.82-1.01; P <0.0005) for all cases and 0.954 (95% confidence interval, 0.881-1.027; P <0.0005) for cases with nondiagnostic serum AFP (<500 microg/L). At a specificity of 90%, the sensitivity was 92%. Both cluster analysis and ANN correctly classified the pooled serum samples, the CLD serum sample with increased AFP, and the HCC patient in complete remission. CONCLUSION: Tumor-specific proteomic signatures may be useful for detection and classification of hepatocellular cancers.

Carcinoma, Hepatocellular↗

PubMed2006

Profiling the cytokines in gingival crevicular fluid using a cytokine antibody array.

BACKGROUND: Various compounds have been detected in gingival crevicular fluid (GCF) as indicators of periodontal disease activity. Therefore, the analysis of GCF may be especially beneficial for diagnosing current periodontal status and addressing the effects of treatment. Moreover, the identification of new markers in GCF may also contribute to elucidating novel mechanisms involved in periodontal disease. This study sought novel marker proteins specific to chronic periodontitis by profiling cytokines in GCF using a cytokine antibody array system. METHODS: Human cytokine array V, which detects 79 cytokines on one membrane, was used to determine the profile of cytokines in GCF from seven subjects with chronic periodontitis and seven subjects with healthy periodontia. The profile was exposed to x-ray film and quantified using image analysis software. Healthy and diseased sites were compared statistically. RESULTS: We detected 10 cytokines in periodontally healthy sites and 36 cytokines in periodontally diseased sites. Interleukin-8 (IL-8) and transforming growth factor-beta 2 (TGF-beta2) were detected at high levels in healthy and diseased subjects. There were significant differences between healthy and diseased subjects in the levels of tissue inhibitor of metalloproteinases-2 (TIMP-2), tumor necrosis factor-beta (TNF-beta), growth-related oncogene (GRO), interferon-inducible protein-10 (IP-10), angiogenin (Ang), vascular endothelial growth factor (VEGF), insulin-like growth factor binding protein-3 (IGFBP-3), osteoprotegerin (OPG), epidermal growth factor (EGF), glial-derived neurotrophic factor (GDNF), pulmonary and activation-regulated chemokine (PARC), oncostatin M (OSM), fibroblast growth factor-4 (FGF-4), IL-16, homologous to lymphotoxins (LIGHT), and placenta growth factor (PlGF). Of these, the newly detected cytokines were GRO, Ang, IGFBP-3, GDNF, PARC, OSM, FGF-4, IL-16, LIGHT, and PlGF. CONCLUSIONS: In this study, we detected several cytokines in GCF using a cytokine antibody array system, including both inflammatory cytokines and various growth factors. Therefore, periodontal disease may participate in the wound healing process and in tissue destruction via the inflammatory process. Our results suggest that the quantification of these cytokines in GCF provides useful information for the diagnosis of periodontal disease status.

Aged↗

PubMed2005

Correlation of bFGF expression in renal cell cancer with clinical and histopathological features by tissue microarray analysis and measurement of serum levels.

The prognostic value of bFGF for surgically treated renal cell cancer (RCC) patients was evaluated by immunohistochemistry (IHC) and the tissue microarray technique (TMA). Additionally, preoperative serum bFGF levels were correlated to tumour stage and the presence of metastases at initial diagnosis. Serum levels of bFGF were measured by ELISA in 39 healthy volunteers, in 37 patients with benign urologic diseases and in 74 RCC patients, 26 of whom revealed lymph node or distant metastases. bFGF expression as detected by IHC was investigated in 777 tissue cores from 259 different RCC patients [median follow-up: 138 (36-240) months]. Eighty eight patients died from tumour progression. For each patient, the TMA slides contained a tissue core from the primary tumour, its invasion front and the normal renal parenchyma. bFGF serum levels were higher in RCC patients vs healthy volunteers (P<0.01) and vs patients with benign urologic diseases (P<0.01). Metastasized patients revealed higher bFGF serum levels than organ-confined specimens (P<0.01). As detected by IHC only increased bFGF expression in the invasion front tissue correlated with the patients' long-term survival (log rank test) (P=0.03). In multivariate analysis regional LN metastases (P<0.01), the histological grading (P<0.01), and an increased bFGF expression in the invasion front (P=0.04) independently predicted the patients' clinical prognosis. Not the expression of bFGF in the primary tumour but in its invasion front reflects the aggressiveness of RCC, hereby indicating a different biological potential within both areas. The value of bFGF serum levels as indicators of systemic tumour dissemination remains to be determined.

Adult↗

PubMed2005

A method for fabricating uni-dsDNA microarray chip for analyzing DNA-binding proteins.

This paper describes an approach for preparing unimolecular double-stranded DNA (uni-dsDNA) microarray chip. In this method, the various target oligonucleotides containing a reverse complementary sequence at 5' end were firstly annealed to a same universal oligonucleotide with amino group at 5' end and immobilized on aldehyde-derivatized glass slide. An on-chip DNA polymerization reaction was then performed to elongate the universal oligonucleotides. After a denaturation and a followed intra-strand annealing, a hairpin structure was formed at the free 3' end of the immobilized oligonucleotides. Finally, another on-chip DNA polymerization was done to synthesize the uni-dsDNA microarray. Combining with a PCR amplification of chemically synthesized target oligonucleotides, this method was much cost-effective for production of the uni-dsDNA microarray. The uni-dsDNA microarray was verified applicable for detecting the presence and monitoring the DNA-binding activity of the sequence-specific DNA-binding proteins.

Cost-Benefit Analysis↗