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Results for “Protein Array Analysis”

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At least 1,135 records · Page 63Linked to original sources

PubMed2006

Analytical and clinical evaluation of a new heart-type fatty acid-binding protein automated assay.

BACKGROUND: The accurate and rapid recognition of myocardial injury in patients presenting in the emergency department (ED) with chest pain continues to be a clinical challenge. Heart-type fatty acid-binding protein (H-FABP) appears to be one of the best candidates among the new early cardiac markers studied. METHODS: We evaluated the analytical characteristics of a new quantitative and fully automated H-FABP assay (Randox Laboratories Ltd., Crumlin, UK) and compared its clinical performance with respect to the myoglobin (Myo) assay (Dade Behring, Milan, Italy). A precision study was carried out by testing three levels of quality control (QC) material and two in-house pool (P) samples. To test the accuracy of H-FABP determinations in plasma (lithium-heparin) samples, H-FABP concentrations measured in a set of matched sera and plasma samples were compared. A total of 77 non-consecutive patients (51 males and 26 females; 62+/-16 years) who presented to the ED with chest pain suggesting myocardial ischemia were enrolled. The patients were classified into two groups (acute myocardial infarction, n=22; non-acute myocardial infarction, n=55) on the basis of the discharge diagnosis. RESULTS: The between-day imprecision for three levels of control material and serum pool samples was 6.26%-8.04% (range 2.32-44.03 microg/L) and 9.03%-12.63% (range 11.85-65.13 microg/L), respectively. In the serum vs. plasma study, bias was +0.178 (95% CI -0.033 to +0.389). The best cut-off and the associated diagnostic efficacy were 95 microg/L and 89.47% for Myo and 5.09 microg/L and 98.70% for H-FABP, respectively. CONCLUSIONS: H-FABP determination in patients with ischemic symptoms may be a more reliable early indication of cardiac damage than myoglobin.

Aged↗

PubMed2004

[Proteome analysis for identification of tumor-associated biomarkers in breast cancer].

BACKGROUND & OBJECTIVE: Pre-symptomatic screening of early-stage breast cancer may greatly reduce tumor-related mortality. Some tumor markers, such as CA15-3 and CA27-29, are recommended only for monitoring therapy of advanced or relapsed breast cancer. This study was to find new biomarkers that could be used individually or in combination with an existing modality for cost-effective screening of breast cancer by proteome analysis. METHODS: Protein expression differences among 128 serum samples of 64 breast cancer patients (19 of stage I, 24 of stage II, and 21 of stage III), 52 patients with benign breast diseases, and 12 healthy women were analyzed with IMAC3 and WCX2 Ciphergen ProteinChip Arrays. RESULTS: On WCX2 chip, a panel of 5 proteins (9 116, 8 905, 8 749, 9 470, and 9 692 Da) was selected based on their collective contribution to the optimal separation between breast cancer patients and both non-cancer patients and healthy women, and expression of another 2 proteins (9 405 and 6 424 Da) was different between patients with breast cancer of stage I and stage III. On IMAC3 chip, a panel of 9 proteins (5 236, 7 823, 7 464, 5 213, 5 334, 5 064, 5 374, 7 756, and 7 623 Da) was selected based on their collective contribution to the optimal separation between breast cancer patients and both non-cancer patients and healthy women, and expression of another 3 proteins (7 922, 4 641, and 5 910 Da) was different between patients with breast cancer of stage I and stage III. CONCLUSION: Protein expression in breast cancer patients is different from that in both non-cancer patients and healthy women, and those proteins with different expression may be used as new biomarkers in breast cancer.

Adult↗

PubMedDate not supplied

Elevated HGF levels in sera from breast cancer patients detected using a protein microarray ELISA.

We developed an ELISA in high-density microarray format to detect hepatocyte growth factor (HGF) in human serum. The microassay can detect HGF at sub-pg/mL concentrations in sample volumes of 100 microL or less. The microassay is also quantitative and was used to detect elevated HGF levels in sera from recurrent breast cancer patients. The microarray format provides the potential for high-throughput quantitation of multiple biomarkers in parallel, as demonstrated with a multiplex analysis of five biomarker proteins.

Biomarkers, Tumor↗

PubMed2005

Controlled protein precipitation in combination with chip-based nanospray infusion mass spectrometry. An approach for metabolomics profiling of plasma.

Liquid chromatography-mass spectrometry (LC-MS) is a common method for profiling biological samples in metabolomics. However, LC-MS data of metabolomic studies are often affected by high noise levels, retention time shifts, and high variability in signal intensities. With a new chip-based nanoelectrospray source it becomes possible to directly infuse complex biological samples such as plasma without any chromatographic separation beforehand. In combination with highly diluted samples and long data acquisition times, the parallel analysis of hundreds of compounds is now possible. In a proof-of-concept study, 10 human plasma samples from females and males were analyzed with the intention to separate the two groups by their different metabolomes. The reproducibility was so high that statistical analysis of the data could be performed without prior normalization. Two groups of female and male samples were separated by a supervised machine learning algorithm, principal component analysis, and hierarchical clustering. Peaks contributing to the group separation were characterized by accurate mass measurement and MS-MS fragmentation and by spiking experiments. The feasibility of direct sample infusion using the new chip-based nanoelectrospray device opens a new dimension for the rapid parallel analysis of complex biological mixtures.

Adult↗

PubMed2005

Automation of biochip array technology for quality results.

BACKGROUND: Proteomics' requirement for simultaneous measurement of multiple markers is now possible with biochip array technology. Many laboratories utilise in-house, manual procedures for biochip fabrication and sample testing. Reproducibility and standardisation of biochip processes is vital to ensure quality of results and offer the best tool for elucidation of complex relationships between multiple proteins in diseased conditions. METHODS: Various novel control checks have been implemented in biochip fabrication, reagent manufacture, automation and imaging processes for the Evidence analyser. Reference spots enable location of discrete test regions on the surface of the biochip and simultaneous quantification of multiple markers. Performance and standardisation methods are presented. RESULTS: Formulation of dispense solution for discrete test regions had a direct effect on their shape, stability and integrity on the biochip surface. Assays for fertility hormones and drugs of abuse demonstrated excellent precision, stability and comparison with other commercial methods. CONCLUSION: Control processes employed in the manufacture and analysis of Evidence components ensure reproducibility of assays for a range of routine and novel markers.

Animals↗

PubMed2004

Protein profiling of cervical cancer by protein-biochips: proteomic scoring to discriminate cervical cancer from normal cervix.

Analysis of multiple proteins is thought to be essential for establishment of signature proteomic patterns that may distinguish cancer from non-cancer. Surface-enhanced laser desorption/ionization (SELDI) is an affinity-based mass spectrometric method in which proteins of interest are selectively absorbed to a chemically modified surface on a biochip. This technology may provide protein profiling of a variety of biological specimens. In this study, we explored whether the protein biochip SELDI approach could differentiate cervical cancer from non-cancer cohorts. We screened protein profiles generated by SELDI in 62 cervical epithelial cell samples microdissected from 35 invasive cervical cancer and 27 age-matched normal cervix tissue specimens, respectively. The cell lysates of pure populations of cervical cells were applied onto Ciphergen ProteinChip WCX2 Arrays. Proteins bound to the chips were analyzed on a ProteinChip Reader Model PBS II. Derived proteomic patterns were converted to a simple proteomic scoring for distinguishing cancer from non-cancer cohorts. SELDI protein profiles of cell lysates from 20 cervical cancer and 15 normal cervix tissue specimens were used to train and develop a classification scoring system that used a seven-protein mass pattern. The training samples could be correctly discriminated. When a test set of 27 samples was used for evaluation of this scoring system to distinguish cervical cancer from non-cancer, a sensitivity of 87%, a specificity of 100%, a positive predictive value of 100%, and a negative predictive value of 86% for the test population were obtained. All seven proteins appeared to be down regulated in cervical cancer. The results from this study indicate that the proteomics approach of SELDI mass spectrometry, in combination with a simple scoring system, may distinguish cervical cancer from its normal counterpart. If this approach is also workable in the analysis of cervical exfoliated cell lysate, it might potentially be used in the early diagnosis of invasive cervical cancer. In addition, the identification of these specific proteins in cervical cancer may also facilitate the discovery of new cervical tumor marker(s).

Adult↗

PubMed2006

Molecular profiling of human endometrium during the menstrual cycle.

This summary of the findings of investigations of the changing transcriptional profile of human endometrium during the menstrual cycle shows that it is possible to classify the menstrual cycle based on the global gene expression profile, and identifies groups of known and novel genes that may be associated with different biological processes that occur in the endometrium such as implantation and menstruation.

Adult↗

PubMed2002

Combinatorial use of mRNA and two-dimensional electrophoresis expression data to choose relevant features for mass spectrometric identification.

It is only recently that quantitative studies of differential proteome analysis (DPA) have become possible. In this paper the issues involved in quantitative DPA are discussed and novel tools to select features for identification by mass spectrometry (MS) are described. The problem of comparing two sets of gels on a global level is explored as well as how to find specific protein features that differentiate two sets of two-dimensional electrophoresis gels. The concept of a 'virtual' gel, derived from gene expression data, is introduced. The virtual gel enables the co-analysis of data from gene and protein expression. We discuss the value of such an approach, and consider what new information can be gained by using gene and protein expression together. These tools are illustrated by analysis of data from tandem gene and protein expression experiments. Features that are highlighted by the above methods are putative candidates for MS identification. Tools are described that integrate the process of feature selection, cutting, and MS analysis.

Breast↗

PubMed2003

Quantitative detection of protein arrays.

We introduce a quantitative method that utilizes scanning electron microscopy for the analysis of protein chips (SEMPC). SEMPC is based upon counting target-coated gold particles interacting specifically with ligands or proteins arrayed on a derivative microscope glass slide by utilizing backscattering electron detection. As model systems, we quantified the interactions of biotin and streptavidin and of an antibody with its cognate hapten. Our method gives quantitative molecule-counting capabilities with an excellent signal-to-noise ratio and demonstrates a broad dynamic range while retaining easy sample preparation and realistic automation capability. Increased sensitivity and dynamic range are achieved in comparison to currently used array detection methods such as fluorescence, with no signal bleaching, affording high reproducibility and compatibility with miniaturization. Thus, our approach facilitates the determination of the absolute number of molecules bound to the chip rather than their relative amounts, as well as the use of smaller samples.

Antibodies↗

PubMed2006

Protein profiling of bladder cancer using the 2D-PAGE and SELDI-TOF-MS technique.

Protein profiling is a promising tool for tumor characterization and the detection of tumor markers in bladder cancer. Techniques for 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and surface-enhanced laser desorption/ionization with time-of-flight mass spectrometry (SELDI-TOF-MS) have improved; both were evaluated using bladder tumor tissue. Normal urothelium and pTa G2, pT1 G3, and >or=pT3 G3 tissues were obtained from the operating room and, after macrodissection, subjected to 2D-PAGE and to SELDI-TOF-MS ProteinChip. 2D-PAGE gels expressed significantly different protein patterns for pTa G2 and pT3 G3 tumors. pT1 G3 tumors showed expression profiles similar to those of the invasive tumors, with upregulation of galectin 3, gelsolin, villin 2, moesin, and annexin 6. Similarly, distinct protein peaks were detected for superficial and muscle-invasive urothelial cancers by SELDI-TOF-MS. Six of seven superficial pTa G2 tumors showed an intense peak at 6.7 and 10.1 kD, while invasive carcinomas showed an intense peak near 9.5 kD. No disturbing influence of surrounding tissue on the results was detected. It was shown that both techniques (2D-PAGE and ProteinChip) work well, and especially ProteinChip analysis seems promising for clinical application.

Biomarkers, Tumor↗

PubMed2004

Development of miniaturized competitive immunoassays on a protein chip as a screening tool for drugs.

BACKGROUND: Doping in sports has become a serious problem. Gas chromatography-mass spectrometry (GC-MS) serves as an effective reference method, but it is limited by low throughput and is therefore not suitable for large-scale screening. Use of protein chips for high-throughput screening of all athletes for prohibited substances could become an important complementary tool to GC-MS. METHODS: We developed a protein chip based on an aldehyde-activated glass slide containing 10 physically isolated arrays. The chip was used to screen urine from 1347 athletes for prohibited substances and to screen a negative control group consisting of 200 females and 120 males. Urine samples from 66 individuals known to be abusers, provided by the China Doping Control Center (CDCC), and 129 standard prohibited substances were tested as positive controls. RESULTS: All 1347 urine samples screened by means of the protein chips were also subjected to reference analysis by GC-MS at the CDCC. There was no qualitative difference between the results obtained with the two methods. The correlation coefficient (r(2)) for the quantitative results obtained with the protein chip and GC-MS was 0.991. CONCLUSIONS: The protein chip could be used to screen for a series of 16 prohibited drugs in urine samples. This system has the potential to become an effective screening method to test substances prohibited by the International Olympic Committee.

Doping in Sports↗

PubMed2006

Effect of antifungal agents on protein composition of Candida albicans studied by capillary electrophoresis and chip technology.

In the present study protein profile of a Candida albicans strain had been examined by chip technology and conventional capillary electrophoresis (CE). Profiles could be characterised by the presence of ten dominating protein peaks. These proteins could be distinguished by both techniques, but their quantity showed significant differences in the electropherograms obtained by CE and chip method. Changes in the protein profile were induced by administration of different antifungal agents. Fluconazole and amphotericin B treatment was able to induce similar changes in the pattern, appearance of a 40-kDa protein and up-regulation of a 60-kDa protein was observed by chip technology. Increase in the quantity of these proteins under stress effect (antifungal treatment) might refer to their stress function in the fungal cell. Treatment of C. albicans cells with MK 94 (fused cyclic Mannich ketone) antifungal compound induced not only the previously mentioned changes, but further specific alterations, appearance of a 19-kDa protein and up-regulation of the low molecular weight proteins. This might refer to the different mode of action of this agent on the fungal cells. Conventional capillary electrophoresis was suitable to detect the appearance of the 19-kDa peak, and up-regulation of the 60 kDa protein, but the other changes could not be detected by this technique. Shorter running time, more effective and baseline separation of proteins refer to the advantages of microchip-based method in the analysis of complex biological samples.

Amphotericin B↗

PubMed2006

Proteomic profiling of accessory structures from the mouse sperm flagellum.

The flagellum of a mammalian spermatozoon consists of an axoneme surrounded in distinct regions by accessory structures known as the fibrous sheath, outer dense fibers, and the mitochondrial sheath. Although the characterization of individual proteins has provided clues about the roles of these accessory structures, a more complete understanding of flagellar function requires the identification of all the polypeptides in these assemblies. Epididymal mouse sperm were treated with SDS to dislodge sperm heads and to extract the axoneme and membranous elements. The remaining flagellar accessory structures were purified by sucrose gradient centrifugation. Analysis of proteins from these structures by two-dimensional gel electrophoresis and colloidal Coomassie Blue staining showed a highly reproducible pattern of >200 spots. Individual spots were picked, digested with trypsin, and identified by mass spectrometry and peptide microsequencing. Approximately 50 individual proteins were identified that could be assigned to five general categories: 1) proteins previously reported to localize to the accessory structures, e.g. ODF2 in the outer dense fibers, the sperm-specific glyceraldehyde-3-phosphate dehydrogenase in the fibrous sheath, and glutathione peroxidase in the mitochondrial sheath, validating this proteomic approach; 2) proteins that had not been shown to localize to any accessory structure but would be predicted to be present, e.g. glycolytic enzymes; 3) proteins known to be part of the flagellum but not localized to a specific site, e.g. adenylate kinase; 4) proteins not expected to be part of the accessory structures based on their previously reported locations, e.g. tektins; and 5) unknown proteins for which no information is available to make a determination as to location. The unexpected presence of the tektins in the accessory structures of the flagellum was confirmed by both immunoblot and immunofluorescence analysis. This proteomic analysis identified a number of unexpected and novel proteins in the accessory structures of the mammalian flagellum.

Animals↗

PubMed2005

Cell biological evaluation of liver cell carcinoma, dysplasia and adenoma by tissue micro-array analysis.

The clinical and morphological definition of hepatocellular carcinoma (HCC), dysplasia and adenoma suffers from a lack of biological understanding. This is especially important in the histomorphological diagnosis of nodular liver lesions in needle biopsies. Therefore, we constructed a liver tissue micro-array (TMA) and evaluated 48 HCCs, 46 dysplasias, 8 adenomas, 20 cirrhotic specimens and 28 normal liver samples derived from 68 patients. Protein (over)expression by tumor suppressor genes p16, p53 and Rb1 was assessed by immunohistochemistry, the proliferative capacity was examined by immunostaining of Ki67. Further, DNA ploidy status (hyperdiploidy) was measured by fluorescent in situ hybridization (FISH) with a chromosome 1-specific repetitive DNA probe. An abnormal chromosome 1 number, i.e. the percentage of hyperdiploid cells, was 11.0, 13.7, 16.1, 23.7 and 31.3 for normal liver samples, adenomas, cirrhosis, dysplasias and HCCs, respectively. A significant difference was found for HCC versus cirrhosis (P = 0.024) or adenoma (P = 0.033), a trend (borderline significance) was seen for dysplasia versus cirrhosis (P = 0.094). Immunohistochemical protein localisation of p53 and Rb1, as well as Ki67 indicating proliferation, was clearly higher in HCC than in cirrhosis or dysplasia (all P < 0.001). Proliferation was also higher in HCC than in adenoma (P = 0.025), whereas a trend (borderline significance) was observed for Rb1 overexpression (P = 0.063). These data suggest that in the liver cell dysplasia-carcinoma pathway, changes in ploidy are followed by increased proliferation and cell biological perturbations involving p53 and Rb1. Adenomas can be distinguished from carcinomas, but not from dysplasias, based on ploidy and proliferation characteristics.

Adenoma, Liver Cell↗

PubMed2002

Improved chip design for integrated solid-phase microextraction in on-line proteomic sample preparation.

A recently introduced silicon microextraction chip (SMEC), used for on-line proteomic sample preparation, has proved to facilitate the process of protein identification by sample clean up and enrichment of peptides. It is demonstrated that a novel grid-SMEC design improves the operating characteristics for solid-phase microextraction, by reducing dispersion effects and thereby improving the sample preparation conditions. The structures investigated in this paper are treated both numerically and experimentally. The numerical approach is based on finite element analysis of the microfluidic flow in the microchip. The analysis is accomplished by use of the computational fluid dynamics-module FLOTRAN in the ANSYS software package. The modeling and analysis of the previously reported weir-SMEC design indicates some severe drawbacks, that can be reduced by changing the microextraction chip geometry to the grid-SMEC design. The overall analytical performance was thereby improved and also verified by experimental work. Matrix-assisted laser desorption/ionization mass spectra of model peptides extracted from both the weir-SMEC and the new grid-SMEC support the numerical analysis results. Further use of numerical modeling and analysis of the SMEC structures is also discussed and suggested in this work.

Miniaturization↗

PubMed2006

High-throughput analysis of mumps virus and the virus-specific monoclonal antibody on the arrays of a cationic polyelectrolyte with a spectral SPR biosensor.

We investigated the potential use of a spectral surface plasmon resonance (SPR) biosensor in a high-throughput analysis of mumps virus and a mumps virus-specific mAb on the arrays of a cationic polyelectrolyte, poly(diallyldimethylammonium chloride) (PDDA). The PDDA surface was constructed by electrostatic adsorption of the polyelectrolyte onto a monolayer of 11-mercaptoundecanoic acid (MUA). Poly-L-lysine was also adsorbed onto the MUA monolayer and compared with the PDDA surface in the capacity of mumps virus immobilization. The PDDA surface showed a higher adsorption of mumps virus than the poly-L-lysine surface. The SPR signal caused by the virus binding onto the PDDA surface was proportional to the concentration of mumps virus from 0.5 x 10(5) to 14 x 10(5) pfu/mL. The surface structure of the virus arrays was visualized by atomic force microscopy. Then, a dose-dependent increase in the SPR signal was observed when various concentrations of the antimumps virus antibody in buffer or human serum were applied to the virus arrays, and their interaction was specific. Thus, it is likely that the spectral SPR biosensor based on the cationic polyelectrolyte surface may provide an efficient system for a high-throughput analysis of intact virus and serodiagnosis of infectious diseases.

Antibodies, Monoclonal↗

PubMed2006

Airway cytokine expression measured by means of protein array in exhaled breath condensate: correlation with physiologic properties in asthmatic patients.

BACKGROUND: Simultaneous monitoring of airway inflammation and physiology might be useful for asthma management. OBJECTIVE: We examined the upregulated molecules in asthmatic airways. Furthermore, we investigated the relationship between these molecules and the airway physiologic properties of asthma. METHODS: Ten nonsmoking healthy subjects and 16 steroid-naive asthmatic patients were enrolled. Exhaled breath condensate (EBC) sampling, spirometry, and methacholine inhalation challenge were performed on one occasion in this cross-sectional study. Peak expiratory flow was also measured for 4 weeks. Airway cytokine-chemokine-growth factor production was analyzed with a protein array. RESULTS: The expressions of IL-4, IL-8, IL-17, TNF-alpha, RANTES, IFN-gamma-inducible protein 10, TGF-beta, and macrophage inflammatory protein 1alpha and 1beta were significantly upregulated in asthmatic airways compared with those of nonsmoking healthy subjects. Among the upregulated molecules, RANTES expression was significantly correlated with the parameters that represent airway caliber, FEV(1) and respiratory resistance values. In addition, the levels of both TNF-alpha and TGF-beta were significantly correlated with the methacholine threshold and peak expiratory flow variability for the week. CONCLUSION: Inflammatory molecule analysis with EBC appeared to be useful for monitoring the asthmatic airway condition. CLINICAL IMPLICATIONS: Measurements of cytokine levels in EBC might be a promising approach to assess the efficacy of pharmacologic interventions and to investigate the pathophysiology of asthma.

Adult↗

PubMed2007

Application of phenylboronic acid modified hydrogel affinity chips for high-throughput mass spectrometric analysis of glycated proteins.

The complexity of the human plasma proteome is greatly increased by post-translational modifications. Besides physiological modifications, pathological conditions such as diabetes are responsible for adding to this complexity by producing advanced glycation endproducts (AGEs). When searching for specific biomarkers it is a prerequisite to reduce this complexity prior to analysis. To do this, agarose hydrogel was used to create a high-capacity affinity layer on the modified aluminum surface of MALDI (matrix-assisted laser desorption/ionization) targets. 3-Aminophenylboronic acid was immobilized via cyanogen bromide activation as a ligand for affinity sorption of glycated proteins, followed by their direct detection by MALDI. High protein capacity of prepared MALDI chips, efficient separation and low non-specific protein binding were demonstrated. The results show that phenylboronic acid modified hydrogels are very suitable for creating affinity surfaces for the high-throughput analysis of AGEs.

Aluminum↗