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PubMedDate not supplied

Comparison of multiplexed techniques for detection of bacterial and viral proteins.

Immobilized antibody microarrays were compared to the Luminex flow cytometry system that utilizes suspensions of polystyrene microbeads covalently coupled with capture antibodies. The two immunoassays were performed for comparison of reproducibility, limits of detection and dynamic range. The Luminex system showed lower limits of detection and increased dynamic range among samples whereas the protein microarrays could be more amenable to miniaturization. Both technologies were capable of sensitive multiplexed detection.

Antibodies, Bacterial↗
PubMed2006

High-throughput screening methodology for the directed evolution of glycosyltransferases.

Engineering of glycosyltransferases (GTs) with desired substrate specificity for the synthesis of new oligosaccharides holds great potential for the development of the field of glycobiology. However, engineering of GTs by directed evolution methodologies is hampered by the lack of efficient screening systems for sugar-transfer activity. We report here the development of a new fluorescence-based high-throughput screening (HTS) methodology for the directed evolution of sialyltransferases (STs). Using this methodology, we detected the formation of sialosides in intact Escherichia coli cells by selectively trapping the fluorescently labeled transfer products in the cell and analyzing and sorting the resulting cell population using a fluorescence-activated cell sorter (FACS). We screened a library of >10(6) ST mutants using this methodology and found a variant with up to 400-fold higher catalytic efficiency for transfer to a variety of fluorescently labeled acceptor sugars, including a thiosugar, yielding a metabolically stable product.

Amino Acid Sequence↗
PubMed2004

Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum.

Two non-cationic defensin-like antimicrobial peptides, named Amblyomma defensin peptide 1 and Amblyomma defensin peptide 2, were identified from the hard tick, Amblyomma hebraeum, by a combination of suppression subtractive hybridization for differentially expressed genes and proteomics. cDNA clones encoding each of these two defensin-like antimicrobial peptides were isolated from the differentially expressed cDNA library of the tick synganglia (central nervous system). The preproproteins deduced from the cDNA sequences each have 92 amino acid residues. Amblyomma defensin peptide 2 was purified from the haemolymph of fed female ticks. The purified peptide displayed antibacterial activity against Gram-negative and Gram-positive bacteria. Amblyomma defensin peptide 1 was further identified by protein chip capture combined with SELDI-TOF (surface-enhanced laser desorption/ionization-time-of-flight) MS. By screening for differentially expressed proteins, it was found that the expression of Amblyomma defensin peptide 1 was upregulated during 4 days post-feeding. Our findings firstly provide two defensin-like antimicrobial peptides that are particularly novel in being anionic, together with corresponding cDNA sequences, in hard ticks, and prove that the combination of suppression subtractive hybridization and protein profiling is a powerful method to study differentially expressed proteins, especially for organisms without available genome sequence information.

Amino Acid Sequence↗
PubMed2003

Microarrayed recombinant allergens for diagnosis of allergy.

We suggest that the coapplication of recombinant allergens and microarray technology can lead to the development of new forms of multi-allergen tests which allow the determining and monitoring of complex sensitization profiles of allergic patients in single assays. The allergen extracts which have so far been used for diagnosis only allowed the determining of whether an allergic patient is sensitized against a particular allergen source, but the disease-eliciting allergens could not be identified. Through the application of recombinant DNA technology a rapidly growing panel of recombinant allergen molecules has become available which meanwhile comprises the epitope spectrum of most of the important allergen sources. We demonstrate that microarray technology can be used to establish multi-allergen tests consisting of microarrayed recombinant allergen molecules. Microarrayed recombinant allergens can be used to determine and monitor the profile of disease-eliciting allergens using single tests that require minute amounts of serum from allergic patients. The wealth of diagnostic information gained through microarray-based allergy testing will likely improve diagnosis, prevention and treatment of allergy.

Allergens↗
PubMed2003

Allergen microarray: comparison of microarray using recombinant allergens with conventional diagnostic methods to detect allergen-specific serum immunoglobulin E.

BACKGROUND: The availability of recombinant allergens and recent advances in biochip technology led to the development of a novel test system for the detection of allergen-specific IgE. OBJECTIVE: To test the performance of this allergen microarray in a serological analytical study. METHODS: Standard allergens contained in grass pollen (Phl p 1, Phl p 2, Phl p 5 and Phl p 6) and tree pollen (Bet v 1 and Bet v 2) were used as a model system. The detection of allergen-specific serum IgE using microarrays was compared with standard test systems: CAP/RAST and an in-house ELISA. In order to test the analytical sensitivity of the assays, geometric dilutions of a serum pool containing high levels of pollen-specific IgE from allergic individuals were tested in each system. To assess the analytical specificity, the sera of 51 patients with presumptive allergic symptoms were collected before diagnosis. Thereafter, the results for grass/tree-pollen-specific IgE were compared. RESULTS: The microarray has a good dynamic range similar to the CAP/RAST system. Microarray and ELISA showed comparable analytical sensitivity exceeding the CAP/RAST system. With respect to the analytical specificity, no significant cross-reactivity of the allergens was observed. For two of the allergens tested, weak positive signals were detected in the microarray test system, whereas they were not detectable by CAP/RAST. CONCLUSION: A good correlation of presently used methods to detect serum IgE and the novel microarray test system was observed. As a next step, a careful validation of this method for a multitude of allergens and a thorough clinical evaluation has to be provided. Microarray testing of allergen-specific IgE can be presumed to be the method of choice for a prospective component-resolved diagnosis of Type I allergy, and the basis for the design and monitoring of a patient-tailored specific immunotherapy in the future.

Allergens↗
PubMed2004

A prototype antibody microarray platform to monitor changes in protein tyrosine phosphorylation.

Reversible protein phosphorylation is a key regulatory process in all living cells. Deregulation of modification control mechanisms, especially in the case of tyrosine, may lead to malignant transformation and disease. Phosphotyrosine (p-Tyr) accounts for only 0.05% of the total cellular phospho-amino acid content, yet plays an unusually prominent role in eukaryotic signaling, development, and growth. Tracking temporal and positional p-Tyr changes across the cellular proteome, i.e. tyrosine phosphoproteomics, is therefore tremendously valuable. Here, we describe and evaluate a prototype antibody (Ab) microarray platform to monitor changes in protein Tyr phosphorylation. Availability permitting, a virtually unlimited number of Abs, each recognizing a specific cellular protein, may be arrayed on a chip, incubated with total cell or tissue extracts or with biological fluids, and then probed with a fluorescently labeled p-Tyr-specific monoclonal Ab, PY-KD1, specifically generated for this assay as part of the current study. The optimized protocol allowed detection of changes in the Tyr phosphorylation state of selected proteins using submicrogram to low nanogram of total protein extract, amounts that may conceivably be obtained from a thousand to a hundred thousand cells, or less, depending on the cell or tissue type. The assay platform was evaluated by assessing changes in a rationally selected subset of the Tyr phosphoproteome of Bcr-Abl-expressing cells treated with a specific inhibitor, Gleevec, and of epidermal growth factor (EGF)-treated HeLa cells. The results, ratiometric rather than strictly quantitative in nature, conformed with previous identifications of several Bcr-Abl and EGF receptor targets, and associated proteins, as detected by exhaustive mass spectrometric analyses. The Ab microarray method described here offers advantages of low sample and reagent consumption, scalability, detection multiplexing, and potential compatibility with microfluidic devices and automation. The system may hold particular promise for dissecting signaling pathways, molecular classification of tumors, and profiling of novel target-cancer drugs.

Animals↗
PubMed2003

Profiling the global tyrosine phosphorylation state.

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome.

Blotting, Far-Western↗
PubMed2005

Robust estimation of protein expression ratios with lysate microarray technology.

MOTIVATION: The protein lysate microarray is a developing proteomic technology for measuring protein expression levels in a large number of biological samples simultaneously. A challenge for accurate quantification is the relatively narrow dynamic range associated with the commonly used chromogenic signal detection system. To facilitate accurate measurement of the relative expression levels, each sample is serially diluted and each diluted version is spotted on a nitrocellulose-coated slide in triplicate. Thus, each sample yields multiple measurements in different dynamic ranges of the detection system. This study aims to develop suitable algorithms that yield accurate representations of the relative expression levels in different samples from multiple data points. RESULTS: We evaluated two algorithms for estimating relative protein expression in different samples on the lysate microarray by means of a cross-validation procedure. For this purpose as well as for quality control we designed a 1440-spot lysate microarray containing 80 identical samples of purified bovine serum albumin, printed in triplicate with six 2-fold dilutions. Our analysis showed that the algorithm based on a robust least squares estimator provided the most accurate quantification of the protein lysate microarray data. We also demonstrated our methods by estimating relative expression levels of p53 and p21 in either p53(+/+) or p53(-/-) HCT116 colon cancer cells after two drug treatments and their combinations on another lysate microarray. AVAILABILITY: http://www.cs.tut.fi/~mirceanc/lysate_array_bioinformatics.htm

Algorithms↗
PubMed2006

Transcription levels of invasion-related genes in prostate cancer cells are modified by inhibitors of tyrosine kinase.

Previous reports have shown that genistein and tyrphostin AG-1478, two tyrosine kinase inhibitors (TKIs), exert multiple cellular effects in prostate carcinoma cells, e.g. a reduction in the production of urokinase plasminogen activator (uPA) and its receptor uPAR, and a decrease in the cells' ability to invade an artificial basement membrane. Microarray technology was used to measure alterations in mRNA levels caused by TKI treatment in two prostatic carcinoma cell lines, PC-3 and DU-145. Genistein treatment led to a reduction of at least 50% in 78 genes in PC-3, while 82 were twofold upregulated. In DU-145, the same treatment resulted in a 50% decreased transcript level in 120 genes, and increased expression in 25 genes. Tyrphostin AG-1478 produced a 50% reduction in mRNA levels in 58 genes in DU-145, whereas no alterations were demonstrated using the tyrphostin in PC-3 cells. Among the effects of TKIs, a lowered uPA and uPAR transcription was demonstrated in genistein-treated cells, while a few metalloproteinases (MMPs) were affected. Transcription of various integrin subunits was also downregulated overall. Several alterations in gene transcription were demonstrated in PC-3 and DU-145 after TKI treatment. This knowledge could be of importance in the search for new therapeutic strategies in prostate cancer treatment, and the interplay between the various effects needs to be investigated further.

Antineoplastic Agents↗
PubMed2006

Upregulation of macrophage migration inhibitory factor and calgizzarin by androgen in TM4 mouse Sertoli cells.

AIM: To identify proteins induced by androgen in Sertoli cells during spermatogenesis. METHODS: We analyzed protein profiles in TM4 Sertoli cells treated with dihydrotestosterone (DHT) using surface enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS). RESULTS: We found increases in the expression of a 5.0-kDa protein at 15 min, an 11.3-kDa protein at 24 h and 4.3 kDa, 5.7 kDa, 5.8 kDa, 9.95 kDa and 9.98 kDa proteins at 48 h after the treatment. In contrast, the expression of 6.3 kDa and 8.6 kDa proteins decreased at 30 min, and 4.9 kDa, 5.0 kDa, 12.4 kDa and 19.8 kDa proteins at 48 h after the treatment. The 11.3-kDa protein was identified as macrophage migration inhibitory factor (MIF) known to having various functions. The 9.98-kDa protein was identified as calgizzarin related to calcium channels. The timing of their expression suggests that MIF and calgizzarin are involved in late regulation of spermatogenesis in Sertoli cells by androgen. CONCLUSION: MIF and calgizzarin are two important androgen-responsive proteins produced by Sertoli cells and they might play a role in regulating spermatogenesis.

Androgens↗
PubMed2004

Examination of human tissue cytosols for expression of sulfotransferase isoform 1A2 (SULT1A2) using a SULT1A2-specific antibody.

Sulfotransferase isoform 1A2 (SULT1A2) is a member of the cytosolic sulfotransferase family of phase II detoxification enzymes. Studies with recombinant enzymes have shown that SULT1A2 can catalyze the bioactivation of several procarcinogens, indicating a potential role in chemical carcinogenesis. However, previous studies have suggested that the SULT1A2 transcript has a splicing defect that might prevent it from becoming translated into protein; therefore, we sought to determine the expression of SULT1A2 in tissues. An antibody directed against a region of human SULT1A2 that differs from other known sulfotransferase isoforms was developed and used to screen a large number of cytosolic fractions from various tissues. Although the SULT1A2 antibody recognized recombinant SULT1A2 and did not cross-react with other SULT isoforms, the expression of SULT1A2 was not detected in any tissue examined. These studies suggest that if SULT1A2 is expressed as protein, the levels are very low and that SULT1A2 probably does not play a physiological role in chemical carcinogenesis.

Amino Acid Sequence↗
PubMed2005

Fully automated synthesis of (phospho)peptide arrays in microtiter plate wells provides efficient access to protein tyrosine kinase characterization.

BACKGROUND: Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Synthetic peptide arrays and libraries, in particular, have accelerated the process. Several factors have hindered or limited the applicability of various techniques, such as the need for deconvolution of combinatorial libraries, the inability or impracticality of achieving full automation using two-dimensional or pin solid phases, the lack of convenient interfacing with standard analytical platforms, or the difficulty of compartmentalization of a planar surface when contact between assay components needs to be avoided. This paper describes a process for synthesis of peptides and phosphopeptides on microtiter plate wells that overcomes previous limitations and demonstrates utility in determination of the epitope of an autophosphorylation site phospho-motif antibody and utility in substrate utilization assays of the protein tyrosine kinase, p60c-src. RESULTS: The overall reproducibility of phospho-peptide synthesis and multiplexed EGF receptor (EGFR) autophosphorylation site (pY1173) antibody ELISA (9H2) was within 5.5 to 8.0%. Mass spectrometric analyses of the released (phospho)peptides showed homogeneous peaks of the expected molecular weights. An overlapping peptide array of the complete EGFR cytoplasmic sequence revealed a high redundancy of 9H2 reactive sites. The eight reactive phospopeptides were structurally related and interestingly, the most conserved antibody reactive peptide motif coincided with a subset of other known EGFR autophosphorylation and SH2 binding motifs and an EGFR optimal substrate motif. Finally, peptides based on known substrate specificities of c-src and related enzymes were synthesized in microtiter plate array format and were phosphorylated by c-Src with the predicted specificities. The level of phosphorylation was proportional to c-Src concentration with sensitivities below 0.1 Units of enzyme. CONCLUSIONS: The ability of this method to interface with various robotics and instrumentation is highly flexible since the microtiter plate is an industry standard. It is highly scalable by increasing the surface area within the well or the number of wells and does not require specialized robotics. The microtiter plate array system is well suited to the study of protein kinase substrates, antigens, binding molecules, and inhibitors since these all can be quantitatively studied at a single uniform, reproducible interface.

Antigen-Antibody Reactions↗
PubMed2005

Population fitness and the regulation of Escherichia coli genes by bacterial viruses.

Temperate bacteriophage parasitize their host by integrating into the host genome where they provide additional genetic information that confers higher fitness on the host bacterium by protecting it against invasion by other bacteriophage, by increasing serum resistance, and by coding for toxins and adhesion factors that help the parasitized bacterium invade or evade its host. Here we ask if a temperate phage can also regulate host genes. We find several different host functions that are down-regulated in lysogens. The pckA gene, required for gluconeogenesis in all living systems, is regulated directly by the principal repressor of many different temperate prophage, the cI protein. cI binds to the regulatory region of pckA, thereby shutting down pckA transcription. The pckA regulatory region has target sequences for many other temperate phage repressors, and thus we suggest that down-regulation of the host pckA pathway increases lysogen fitness by lowering the growth rate of lysogens in energy-poor environments, perhaps as an adaptive response to the host predation system or as an aspect of lysogeny that must be offset by down-regulating pckA.

Bacteriophage lambda↗
PubMed2005

Absolute quantitation of cancer-related proteins using an MS-based peptide chip.

New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific tumor biomarkers and their level of posttranslational modifications. Quantitative molecular profiling of cancer signaling networks would provide a more detailed understanding of the contribution of protein expression and posttranslational modification levels to tumorigenesis. We have developed a unique approach for absolute quantitation of protein expression that integrates affinity capture of proteolytic peptides with mass spectrometry and thus provides detection, identification, and quantitation of their cognate proteins. We have previously shown the high sensitivity and specificity of this approach. Here we demonstrate the absolute quantitation of a model peptide using our technology. We have used this approach to capture epitope-containing peptides from proteolytically digested target proteins, including p53, epidermal growth factor receptor (EGFR), and prostate-specific antigen (PSA). Our technology can easily be extended to the absolute quantitation of protein modification levels, in addition to the determination of protein expression levels, and can be readily adapted for use in a microarray format. This method offers an improved approach to protein chip technology that should prove useful for clinical diagnosis and drug development applications.

Antibodies, Monoclonal↗
PubMed2005

Activation status of the JAK/STAT3 pathway in mantle cell lymphoma.

CONTEXT: Signal transducer and activator of transcription 3 (STAT3) is oncogenic, and we previously found evidence of constitutive STAT3 activation in a relatively small number of frozen mantle cell lymphoma (MCL) cell tumors. OBJECTIVES: To comprehensively survey the activation and phosphorylation status of STAT3 in MCL and to assess if STAT3 activation in these tumors is due to cytokine stimulation by examining the phosphorylation and activation status of Janus kinase (JAK), the physiologic activator of STAT3. DESIGN: We evaluated 43 formalin-fixed, paraffin-embedded MCL tumors using immunohistochemistry and phospho-specific antibodies against STAT3 and JAK. RESULTS: There were 37 small cell and 6 blastoid cases. There was heterogeneous expression of phospho-STAT3 (pSTAT3), with 23 negative cases (53%), 12 weakly positive cases (28%), and 8 strongly positive cases (19%). JAK3 was the only member detectable in 3 MCL cell lines, and immunoprecipitation data showed a relatively low level of tyrosine phosphorylation of JAK3 in these cells. Using immunohistochemistry, phospho-JAK3 (pJAK3) was detectable in 18 (44%) of 41 MCL tumors examined, and pJAK3 expression correlated with that of pSTAT3 (P = .008). A notable exception to this correlation was seen in the blastoid variant, since 4 (67%) of 6 blastoid cases were pSTAT3 positive but pJAK3 negative. CONCLUSIONS: We have confirmed our previous finding that STAT3 is constitutively activated in MCL tumors, with an overall frequency of 47% in this series. STAT3 activation in the small cell but not the blastoid variant of MCL is likely mediated by JAK3.

Adult↗
PubMed2005

Protein expression profiling identifies subclasses of breast cancer and predicts prognosis.

Breast cancer is a heterogeneous disease whose evolution is difficult to predict by using classic histoclinical prognostic factors. Prognostic classification can benefit from molecular analyses such as large-scale expression profiling. Using immunohistochemistry on tissue microarrays, we have monitored the expression of 26 selected proteins in more than 1,600 cancer samples from 552 consecutive patients with early breast cancer. Both an unsupervised approach and a new supervised method were used to analyze these profiles. Hierarchical clustering identified relevant clusters of coexpressed proteins and clusters of tumors. We delineated protein clusters associated with the estrogen receptor and with proliferation. Tumor clusters correlated with several histoclinical features of samples, including 5-year metastasis-free survival (MFS), and with the recently proposed pathophysiologic taxonomy of disease. The supervised method identified a set of 21 proteins whose combined expression significantly correlated to MFS in a learning set of 368 patients (P < 0.0001) and in a validation set of 184 patients (P < 0.0001). Among the 552 patients, the 5-year MFS was 90% for patients classified in the "good-prognosis class" and 61% for those classified in the "poor-prognosis class" (P < 0.0001). This difference remained significant when the molecular grouping was applied according to lymph node or estrogen receptor status, as well as the type of adjuvant systemic therapy. In multivariate analysis, the 21-protein set was the strongest independent predictor of clinical outcome. These results show that protein expression profiling may be a clinically useful approach to assess breast cancer heterogeneity and prognosis in stage I, II, or III disease.

Adult↗
PubMed2003

Current status of protein chip development in terms of fabrication and application.

Sequencing of the human genome revealed that more than 30 000 genes encode proteins comprising the human proteome. "Proteomics" can be defined as a field of research studying proteins in terms of their function, expression, structure, modification and their interaction in physiological and in pathological states. The concentration, modification and interaction of proteins in cells, plasma, and in tissues are crucial in determining the phenotype of living organisms. Although fluctuation of protein concentration is essential to maintain homeostasis, protein expression levels are also pathognomonic features. Estimating protein concentration by analyzing the quantity of mRNA in cells through conventional technologies, such as DNA chips, does not provide precise values since the half-life and translation efficacy of mRNA is variable. In addition, polypeptides undergo post-translational modification. For these reasons, novel techniques are needed to analyze multiple proteins simultaneously using protein microarrays. In the near future, protein chips may allow construction of complete relational databases for metabolic and signal transduction pathways. This article reviews the current status of technologies for fabricating protein microarrays and their applications.

Biotin↗