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Results for “multiplexed proteomics”

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PubMed2004

Protein microarrays and multiplexed sandwich immunoassays: what beats the beads?

Protein microarray technology allows the simultaneous determination of a large variety of parameters from a minute amount of sample within a single experiment. Assay systems based on this technology are currently applied for the identification, quantitation and functional analysis of proteins. Protein microarray technology is of major interest for proteomic research in basic and applied biology as well as for diagnostic applications. Miniaturized and parallelized assay systems have reached adequate sensitivity and hence have the potential to replace singleplex analysis systems. However, robustness and automation needs to be demonstrated before this technology will finally prove suitable for high-throughput applications. Miniaturized and parallelized sandwich immunoassays are the most advanced assays formats among the different protein microarray applications. Multiplexed sandwich immunoassays can be used for the identification of biomarkers and the validation of potential target molecules. In this review an overview will be given on the current stage of protein microarray technology with a special focus on miniaturized multiplexed sandwich immunoassays.

Automation↗

PubMed2006

Biotechnology offers revolution to fish health management.

Biotechnology has many applications in fish health management. The application of monoclonal antibodies (mAbs) provides a rapid means of pathogen identification; antibodies to immunoglobulins from different fish species can be used to monitor the host response following vaccination; and mAbs also have the potential for screening broodstock for previous exposure to pathogens. Luminex technology exemplifies a novel antibody-based method that can be applied to both pathogen detection and vaccine development. Molecular technologies, such as the polymerase chain reaction (PCR), real time PCR and nucleic acid sequence-based amplification (NASBA), have enabled detection, identification and quantification of extremely low levels of aquatic pathogens, and microarray technologies offer a new dimension to multiplex screening for pathogens and host response. Recombinant DNA technology permits large-scale, low-cost vaccine production, moreover DNA vaccination, proteomics, adjuvant design and oral vaccine delivery will undoubtedly foster the development of effective fish vaccines in the future.

Animals↗

PubMed2005

Reverse-phase protein microarrays for tissue-based analysis.

The deciphering of the human genome has elucidated our biological structural design and has generated insights into disease development and pathogenesis. At the same time, knowledge of genetic changes during disease processes has demonstrated the need to move beyond genomics towards proteomics and a systems biology approach to science. Analyzing the proteome comprises more than just a numeration of proteins. In fact, it characterizes proteins within cells in the context of their functional status and interactions in their physiological micro- and macroenvironments. As dysregulated signaling often underpins most human diseases, an overarching goal of proteomics is to profile the working state of signaling pathways, to develop 'circuit maps' of normal and diseased protein networks and identify hyperactive, defective or inoperable transduction pathways. Reverse-phase protein microarrays represent a new technology that can generate a multiplex readout of dozens of phosphorylated events simultaneously to profile the state of a signaling pathway target even after the cell is lyzed and the contents denatured.

Animals↗

PubMed2001

High-throughput peptide identification from protein digests using data-dependent multiplexed tandem FTICR mass spectrometry coupled with capillary liquid chromatography.

Tandem mass spectrometry (MS/MS) plays an important role in the unambiguous identification and structural elucidation of biomolecules. In contrast to conventional MS/MS approaches for protein identification where an individual polypeptide is sequentially selected and dissociated, a multiplexed-MS/MS approach increases throughput by selecting several peptides for simultaneous dissociation using either infrared multiphoton dissociation (IRMPD) or multiple frequency sustained off-resonance irradiation (SORI) collisionally induced dissociation (CID). The high mass measurement accuracy and resolution of FTICR combined with knowledge of peptide dissociation pathways allows the fragments arising from several different parent ions to be assigned. Herein we report the application of multiplexed-MS/MS coupled with on-line separations for the identification of peptides present in complex mixtures (i.e., whole cell lysate digests). Software was developed to enable "on-the-fly" data-dependent peak selection of a subset of polypeptides from each FTICR MS acquisition. In the subsequent MS/MS acquisitions, several coeluting peptides were fragmented simultaneously using either IRMPD or SORI-CID techniques. The utility of this approach has been demonstrated using a bovine serum albumin tryptic digest separated by capillary LC where multiple peptides were readily identified in single MS/MS acquisitions. We also present initial results from multiplexed-MS/MS analysis of a D. radiodurans whole cell digest to illustrate the utility of this approach for high-throughput analysis of a bacterial proteome.

Amino Acid Sequence↗

PubMedDate not supplied

New tumor markers: CA125 and beyond.

A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

Biomarkers, Tumor↗

PubMed2006

Proteomic analysis of secreted proteins in early rheumatoid arthritis: anti-citrulline autoreactivity is associated with up regulation of proinflammatory cytokines.

OBJECTIVES: To identify peripheral blood autoantibody and cytokine profiles that characterise clinically relevant subgroups of patients with early rheumatoid arthritis using arthritis antigen microarrays and a multiplex cytokine assay. METHODS: Serum samples from 56 patients with a diagnosis of rheumatoid arthritis of <6 months' duration were tested. Cytokine profiles were also determined in samples from patients with psoriatic arthritis (PsA) and ankylosing spondylitis (n = 21), and from healthy individuals (n = 19). Data were analysed using Kruskal-Wallis test with Dunn's adjustment for multiple comparisons, linear correlation tests, significance analysis of microarrays (SAM) and hierarchical clustering software. RESULTS: Distinct antibody profiles were associated with subgroups of patients who exhibited high serum levels of tumour necrosis factor (TNF)alpha, interleukin (IL)1beta, IL6, IL13, IL15 and granulocyte macrophage colony-stimulating factor. Significantly increased autoantibody reactivity against citrullinated epitopes was observed in patients within the cytokine "high" subgroup. Increased levels of TNFalpha, IL1alpha, IL12p40 and IL13, and the chemokines eotaxin/CCL11, monocyte chemoattractant protein-1 and interferon-inducible protein 10, were present in early rheumatoid arthritis as compared with controls (p<0.001). Chemokines showed some of the most impressive differences. Only IL8/CXCL8 concentrations were higher in patients with PsA/ankylosing spondylitis (p = 0.02). CONCLUSIONS: Increased blood levels of proinflammatory cytokines are associated with autoantibody targeting of citrullinated antigens and surrogate markers of disease activity in patients with early rheumatoid arthritis. Proteomic analysis of serum autoantibodies, cytokines and chemokines enables stratification of patients with early rheumatoid arthritis into molecular subgroups.

Adult↗

PubMed2026

Comparison of endothelin-1 levels in plasma from human coronary arteries measured by enzyme linked immunosorbent assay and Olink high-throughput proteomics platform.

Endothelin-1 (ET-1) antagonists are increasingly being approved for new treatments for cardiovascular disease, where elevated ET-1 levels contribute to increased vasoconstriction. Further therapeutic targets, including coronary artery disease, are under investigation. The Olink Explore 3072 Proximity Extension Assay platform enables multiplexed high-throughput measurement of ~3000 plasma proteins, from minimal (≤6 µL) sample volumes. However, it is not known if the two oligonucleotide-tagged antibodies raised against preproET-11-212, used in this Olink assay, specifically measure biologically active ET-1 or the other inactive EDN1-encoded peptides, also secreted by human endothelial cells. Paired plasma samples from 29 patients with coronary artery disease were obtained, using a specialised intra-coronary sampling catheter, designed to obtain site specific biochemical information from within coronary arteries. We compared ET-1 concentrations measured with an ET-1 specific ELISA, demonstrated to have no cross-reactivity with other EDN1-encoded peptides versus values obtained using Olink Explore platform. Olink-measured ET-1 correlated significantly with ELISA-derived ET-1 levels (r = 0.53, p = 0.003), and Olink values predicted ELISA results. Olink ET-1 concentrations also correlated with ETB receptor levels (r = 0.40, p < 0.05). These findings indicate that the Olink Explore platform can detect relative changes in biologically active ET-1, supporting its use as a biomarker tool in clinical and translational studies.

Humans↗

PubMed2004

Microarray profiling of antiviral antibodies for the development of diagnostics, vaccines, and therapeutics.

Multiplex analysis of antiviral antibody (Ab) responses provides a potentially powerful strategy for viral diagnosis, prognostication, and development of vaccines and prophylactic Abs. In the coming years, advancements in proteomic technologies will provide even more robust methods to characterize antiviral Ab responses. Biomedical researchers will be faced with the exciting challenge of identifying antiviral Ab specificities that correlate with improved outcomes and efficacious interventions, and translating the findings into more effective diagnostics, prophylactics, and therapeutics.

Animals↗

PubMed2026

RNF43 Mutations Are Associated With the Classical Molecular Subtype, Vigorous Antitumor Immune Responses, and Prolonged Survival in Pancreatic Adenocarcinoma.

RNF43 mutations were correlated with microsatellite status in colorectal cancer and with fewer and later recurrences in pancreatic ductal adenocarcinoma (PDAC). Here, we undertake a detailed assessment of RNF43 mutations in PDAC. A total of 313 PDACs (308 microsatellite stable [MSS] and 5 microsatellite-instable [MSI] cases) underwent next-generation sequencing (Oncomine Tumor Mutation Load assay; Thermo Fisher). Spatial analyses (NanoString) classified PDACs according to their transcriptomic and proteomic immune signaling. Fluorescent imaging was used to define spatial compartments (tumor: pancytokeratin+/CD45- and leukocytes: pancytokeratin-/CD45+). Each of 20 PDACs with RNF43 mutations (RNF43mut) and without RNF43 mutations (RNF43wt) underwent multiplex immunofluorescence analysis to determine immune status. A total of 153 PDACs (22 RNF43mut and 131 RNF43wt cases) underwent bulk RNA sequencing to assign into molecular subtypes. Overall, 24 RNF43 mutations were identified (22 MSS PDACs and 2 MSI PDACs). The incidence of RNF43 mutations in MSS PDACs (7.1%) was consistent with The Cancer Genome Atlas (6.7%). However, RNF43 mutations were more frequent among MSI PDACs (40%). Additionally, RNF43mut had differential frequencies of other mutations (including Wnt pathway genes), higher tumor mutational burden values (5.5 mut/mb vs 1.67 mut/mb; P < .01), and significantly longer overall survival (47 vs 18 months; P < .0001) than RNF43wt. Moreover, RNF43mut exhibited significantly higher densities of CD8+ T lymphocytes, dendritic cells, and B lymphocytes (P < .001) and an upregulation of ITGAX, CD11c, CD8, and HLA-DR compared with RNF43wt. Patients with RNF43mut PDACs were more often of the classical molecular subtype (20/22, 90.9%). RNF43mut PDACs showed high tumor mutational burden values, suggesting increased neoantigen load coupled with an abundance of antigen-presenting immune cells and an upregulation of immune determinants promoting antigen presentation. All this contributes to stronger antitumor immune responses and improved clinical outcomes.

Humans↗

PubMed2006

Quantitative intracellular molecular profiling using a one-dimensional flow system.

We report on the development of one-dimensional microfluidic bead arrays for rapid and quantitative molecular profiling of human cancer cells. This new bioanalytical platform integrates the rapid binding kinetics of suspension bead carriers, the multiplexing and encoding capabilities of gene/protein chips, and the liquid handling advantages of microfluidic devices. Using antibody-conjugated beads in a two-site "sandwich" format, we demonstrate that the proteomic contents of as few as 56 human lung epithelial cancer cells can be determined with high sensitivity and specificity. The results indicate that each cell contains approximately 6 x 10(5) copies of the tumor suppressor protein P53. We have further examined the expression changes of P53, c-Myc, and beta-Actin as a function of anticancer drug treatment and have validated these changes by using Western blotting. This ability to quantitatively analyze normal and diseased cells raises new possibilities in studying cancer heterogeneity and circulating tumor cells.

Animals↗

PubMed2006

Personalized medicine and development of targeted therapies: The upcoming challenge for diagnostic molecular pathology. A review.

Due to continuous technical developments and new insights into the high complexity of many diseases, molecular pathology is a rapidly growing field gaining center stage in the clinical management of tumors as well as in the pharmaceutical development of new anti-cancer drugs. The application of novel compounds in clinical trials has revealed promising results; however, the current diagnostic procedures available for determining which patients will primarily benefit from rational tumor therapy are insufficient. To read a patient's tissue as "deeply" as possible, in the future, gaining information on the morphology and on genetic, proteomic, and epigenetic alterations will be the upcoming task of surgical pathologists experienced in molecular diagnostics to provide the clinicians with information relevant for an individualized medicine. Among the different high-throughput technologies, DNA microarrays are now the first array approach close to enter routine diagnostics. Technically advanced and well-established microarray platforms can nowadays be evaluated by distinct bioinformatic tools capable of identifying both novel genes associated with disease development and clusters of genes predicting clinical outcome of an individual tumor. The automatic, highly parallel analysis of proteins and complex proteins lysates for early detection of cancers such as breast, prostate and ovary as proteomic patterns in the serum also appears at the horizon. In addition, an improved analysis of tumor samples via antibody or reverse-phase protein arrays is likely to provide the pathologist in the future with information about activated oncogenic signaling pathways and other cell functions, such as drug response or the potential to metastasize. While expression microarrays and proteomic analysis rely on relatively unstable material incompatible with paraffin-embedded tissue samples, an investigation of DNA methylation using specialized high-throughput platforms has revealed the potential of being used in future diagnostics. Each of these approaches on its own might not suffice to extract all information required for an efficient individualized diagnostics. Therefore, a "multiplex approach" combining the different biological levels DNA, RNA, and protein, may be necessary to functionally classify malignant tumors. This appears to become a major challenge for diagnostic pathologists.

Drug Design↗

PubMed2004

Clinical proteomics and biomarker discovery.

Early detection of disease generally provides much-improved outcomes by a definitive medical procedure or through lifestyle modification along with specific medical management strategies. For serum biomarkers, which are central to the diagnosis of many diseases, to become truly useful sentinels of pathogenesis, their sensitivity and specificity in both early detection and recurrence monitoring must be improved. Currently, the detection and monitoring of disease markers is based on solitary proteins, and this approach is not always reliable. New classes of biomarkers derived from mass spectroscopy analysis of the low molecular weight proteome have shown improved abilities in the early detection of disease and hence in patient risk stratification and outcome. The development of a modular platform technology with sufficient flexibility and design abstractions allowing for concurrent experimentation, test, and refinement will help speed the progress of mass spectroscopy-derived proteomic pattern-based diagnostics from the scientific laboratory to the medical clinic. For acceptance by scientists, physicians, and regulatory personnel, new bioinformatic tools are essential system components for data management, analysis, and intuitive display of these new and complex data. Clinically engineered mass spectroscopy systems are essential for the further development and validation of multiplexed biomarkers that have shown tremendous promise for the early detection of disease.

Biomarkers↗

PubMed2005

Mass spectrometry-based peptide quantification: applications and limitations.

In various areas of research, proteomics and particularly the quantification of proteins and peptides renders a useful addition to biochemical experiments. The range of possible applications varies from supervision of concentration changes of relevant proteins during biogenesis to differential proteomics approaches, distinguishing, for instance, healthy and diseased states. Furthermore, mass spectrometry-based peptide quantification yields the possibility of using highly sensitive bottom-up approaches for determination of protein regulations as well as multiplexing capability. Thereby, changes in protein abundances may be linked to specific cellular states bearing the opportunity to reveal marker proteins for several diseases.

Amino Acids↗

PubMed2005

Pathology of the future: molecular profiling for targeted therapy.

Recent evidence suggests that each patient's cancer has a unique subset of molecular pathogenetic derangements. These derangements may both genetic and proteomic alterations. Genomic and proteomic research tools enable genome-wide assessment of gene expression as well as kinase driven cell signaling events. These tools are illuminating the molecular derangements of individual tumors, even if these tumors have similar morphological characteristics. A combination of laser capture microdissection with multiplexed phosphoproteomic analysis using reverse phase protein microarray technology is being used to identify protein molecular signatures of individual tumors. The in vivo state of multiple kinase driven signal pathways may be evaluated by reverse phase protein microarray with a panel of specific antibodies developed based upon our knowledge of biological processes. Molecular profiling of individual patient's tumors is currently being evaluated in clinical trials at the National Institutes of Health, National Cancer Institute for monitoring Epidermal Growth Factor (EGF) cell signaling events for patients with breast and ovarian cancer.

Forecasting↗

PubMed2005

Proteomic: new advances in the diagnosis of rheumatoid arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease affecting the joints. A number of novel treatment modalities have been introduced over the past years, and rheumatologists are now attempting to institute optimal treatment in recent-onset arthritis. To facilitate diagnosis during the early stages of disease, when often not all clinical symptoms are manifest, a good serological marker is needed. METHODS: Antibodies directed to citrullinated proteins provide this ability. The most sensitive assay for detecting these antibodies is the so-called anti-cyclic citrullinated peptide, second generation (CCP II) enzyme-linked immunosorbent assay (ELISA). RESULTS: The diagnostic and prognostic potential of anti-CCP antibodies and the availability of a fully automated assay method lead us to conclude that the test is satisfactory for routine use as a serological marker of RA. In addition, we consider the potential of multiplex autoantibody assays, including miniaturized, high-throughput microarray technology, to improve diagnosis and prognostication in early onset arthritis patients.

Arthritis, Rheumatoid↗

PubMed2006

Nanotechnologies for biomolecular detection and medical diagnostics.

Nanotechnology-based platforms for the high-throughput, multiplexed detection of proteins and nucleic acids in heretofore unattainable abundance ranges promise to bring substantial advances in molecular medicine. The emerging approaches reviewed in this article, with reference to their diagnostic potential, include nanotextured surfaces for proteomics, a two-particle sandwich assay for the biological amplification of low-concentration biomolecular signals, and silicon-based nanostructures for the transduction of molecular binding into electrical and mechanical signals, respectively.

Biopolymers↗

PubMed2025

The Proteomic Landscape of CTNNB1 Mutated Low-Grade Early-Stage Endometrial Carcinomas.

Endometrial carcinoma is the most frequent gynecologic malignancy in western countries. In recent years, mutations in CTNNB1 have been associated with worse prognosis in low-risk carcinomas. However, there is a lack of understanding of the proteomic implications of CTNNB1 mutations in this type of tumor. In this study, we performed shotgun proteomics using Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of CTNNB1 mutated and wild-type low-risk endometrial carcinomas. A publicly available proteomic and transcriptomic database was used to validate results. Differential protein expression and Gene Set Enrichment Analysis revealed dysregulation of pathways associated with cell keratinization, immune response modulation, and intracellular calcium regulation. CTNNB1 mutated tumors showed immune dysregulation at multiple levels including cytokine secretion, cell adhesion, and lymphocyte activation. These results were supported by tissue multiplex immunofluorescence analysis, demonstrating reduced CD8 tumor-infiltrating lymphocytes and different immune spatial interaction patterns. Intracellular calcium dysfunction was associated with key transcript dysregulation. We found an increased expression of CAMK2A and ROR2, suggesting a potential role for non-canonical Wnt pathway activation in CTNNB1 mutated tumors.

Humans↗

PubMed2003

Epitope-targeted proteome analysis: towards a large-scale automated protein-protein-interaction mapping utilizing synthetic peptide arrays.

We describe the development of a process for the genome-wide mapping of interactions between protein domains and peptide ligands entirely based on high-throughput biochip technologies. A phage library displaying protein domains from a randomly fragmented and cloned cDNA library will be "panned" on an array of synthetic peptide ligands. After multiplexed affinity enrichment, peptide-specific phage populations will be automatically eluted, propagated, labelled and identified by hybridisation to a DNA microarray. Peptide arrays are synthesized in situ by SPOT synthesis on a planar substrate. By utilizing a commercially available library of human brain cDNA plus a set of distinct model domains cloned into T7-phage, we could show that a single panning round on an array of known peptide ligands for these model domains synthesized on a cellulose membrane can yield an enrichment of better than a factor of 1,000. This is sufficient to detect peptide-specific enrichment of Cy3(post-panning) against Cy5(pre-panning)-labelled phage DNA inserts on a cDNA microarray. Thus, the proof-of-principle of our approach could be successfully demonstrated and first interaction data are being collected.

Amino Acid Sequence↗