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May 1, 2013 (Vol. 33, No. 9)

Metabolomics Advances Reshape Dx Arena

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    The Basic IROA® protocol (A, B, C, and D) grows two populations of isotopically labeled cells, one of which is treated experimentally and the other held as a control (B). When pooled cells are processed the signals for the compounds from both the control and experimental cells may be distinguished (C) and differences between the ratio of their areas are directly indicative of the ratio of the respective sizes of their metabolic pools (D). Outliers to the normalized ratios are metabolic pools that are impacted by the experimental treatment. The Phenotypic IROA protocol (steps E, F, C, D) introduces an IROA standard into a nonlabeled sample or biopsy (E & F). The resulting pooled sample is analyzed using the same steps (C and D) as basic IROA. Basic IROA is fundamentally an unbiased analysis, while the Phenotypic IROA is a targeted analysis for a very large number of compounds. Basic IROA peaks (G) and Phenotypic peaks (H) are readily discriminated. [NextGen Metabolomics]

    Metabolomics analyzes and quantifies all metabolites in a given biological sample. Powerful instrumentation along with sophisticated chemometric software allows for comparison of changes in thousands of chemical entities.

    Metabolomics is becoming sufficiently mature to complement other systems biology tools and to deliver medically relevant results.

    According to some metabolomics researchers, continued development of metabolomics platforms and technologies will inevitably lead to development of rapid and comprehensive disease diagnostics. This and other ideas were discussed at OMICS group’s “Metabolomics and Systems Biology” conference last month.

    “Interpretations of metabolomics signatures will make rapid bedside diagnostics possible,” agreed Chris Beecher, Ph.D., CSO, NextGen Metabolomics. “In 5–10 years this information will transform our diagnostic capabilities. The meaningful metabolome interpretation will be available to a physician within minutes for a fraction of the cost of today’s analyses.”

    NextGen is contributing to this vision by perfecting Isotopic Ratio Outlier Analysis™ (IROA), a mass-spectrometry-based protocol that enables efficient identification of all biological metabolites in a sample and their relative concentration, said Dr. Beecher.

    The technology has its roots in a physiochemical phenomenon of naturally occurring C13 atoms. C13 has an extra neutron producing a pattern of additional peaks for each metabolite in a sample. Because of their low natural abundance and the presence of other elemental isotopes, these additional peaks (called M+1, M+2, etc.) are very small and not very informative.

    “We simply boosted the C13 concentration by growing cells in a culture media engineered with precise balances of C13-bearing components,” continued Dr. Beecher. “When you compare a culture grown with 5% C13 with culture grown in 95% C13, each metabolite will be represented by a symmetrical pattern of C12 and C13 peaks. The parameters of this pattern tell us how many carbons the molecule contains and its mass.”

    The mirror symmetry of MS peaks in IROA experiments enables rapid classification of all peaks as real cell products (have mirror counterparts) or artifacts and contaminants (no enhanced M+1, M+2, etc.). NextGen computer algorithms look for the same symmetry to perform phenotyping experiments.

    To identify metabolites in tissue biopsies, the tissue cells are analyzed together with “standard” cells that were isotopically labeled with IROA C13 media. The tissue-derived metabolites can be easily identified by their mass and position relative to the standard.

    The company demonstrated a significant proof of principle for toxicology analysis of drug candidates. Dr. Beecher’s team used IROA to show the response of every metabolite in the cell to flucytosine, a well-characterized inhibitor of DNA synthesis. The data showed clear response in metabolic pathways related to nucleotide biosynthesis, but not in other pathways.

    “We plan to perfect this technology to answer fundamental questions in drug development,” commented Dr. Beecher. “What toxicities should we expect? What is the biological reason for this toxicity?” NextGen plans to set up internal biomarker and toxicology discovery departments based on IROA technology.

  • Global Signaling Pathway Analysis

    Antibody-based proteomic methods deploy sample fractionation followed by separation by liquid chromatography (LC) and mass spectrometry (MS). Such enrichment strategies lower the “noise” from abundant proteins and focus the instrument time on the proteins of interest.

    “Our motif antibodies recognize a particular post-translational modification (PTM), such as phosphorylation or ubiquitination,” said Matthew P. Stokes, Ph.D., scientist III, proteomics service group, Cell Signaling Technology.

    “Motif antibodies and other generalized enrichment strategies produce large datasets but sometimes miss critical signaling proteins that are without the targeted motif or that are present at low levels. Our PTMScan™ Direct approach is unique; in addition to a PTM, our antibodies recognize the surrounding amino-acid context.”

    Cell Signaling Technology generates hundreds of proprietary antibodies against specific post-translationally modified peptide targets in selected signaling pathways. Antibodies are formulated into one of the six PTMScan Direct Reagents. The DNA Damage/Cell Cycle Reagent, for instance, targets 263 sites on 137 proteins, and the Apoptosis/Autophagy Reagent targets 175 sites on 100 proteins.

    “Each target peptide is rigorously validated using multiple strict criteria,” continued Dr. Stokes. “Moreover, we digest the starting material with trypsin, which helps to expose the binding site and ensures that single peptides are immunoprecipitated rather than whole proteins or protein complexes, further improving specificity of binding to our reagents. Our antibodies are capable of recognizing individual activation sites on a single protein.”

    The company provides custom services for targeted screening and quantification of signaling pathways critical for drug and biomarker discovery. Added Dr. Stokes, “We are constantly innovating and developing new reagents and kits for our customers. But we also maintain an extensive R&D program identifying disease drivers in cancer.”

    The company used its profiling technologies to demonstrate the aberrant activation of anaplastic lymphoma kinase (ALK) in ovarian cancer. The screening data was supported by further biological validation confirming ALK as a potential therapeutic target.

    Further investigations shed light on the underlying mechanisms of acquired resistance to treatment with ALK tyrosine kinase inhibitors. PTMScan Direct Profiling of drug-resistant tumors revealed aberrations in multiple signaling pathways, providing the evidence for rational selection of combination therapies.


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