Recent advances in metabolomics technologies, especially those that combine the complementarily of mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR) enables rapid analysis of thousands of peaks representing hundreds of metabolites. Yet, key barriers remain for large-scale metabolomics applications, most notably: even higher throughput with robust metabolite assignment and quantification;identification of unknown metabolites;analysis of low abundance/labile metabolites;and reconstruction of metabolic networks and regulation. Here, we propose an integrated approach that uses chemoselective (CS) tagging of key metabolite functional groups to boost the speed and accuracy of metabolite identification and enhance detection. Such tagged metabolites are inherently amenable for multiplexed analysis and reliable relative quantification via stable isotope encoding. This approach will accelerate analytical throughput while widening the classes of analytes, including metabolically enriched isotopologues, far beyond current limits. We will achieve our goals via the following specific aims: SA1. To develop CS probes for tagging metabolites by targeting functional groups: Hydrophobic quaternary ammonium (QA)-based CS probes will be developed for tagging carbonyl, amino, thiol, &diol functional groups (FG) in metabolites, optimized for direct infusion FT- ICR-MS detection &assignment;SA2. To develop a set of isotope-encoded CS probes for metabolite identification and multiplexed quantification by FT-ICR-MS and NMR: Developing 13C-encoded QA-CS probes will facilitate integrated structural analysis of metabolites by 13C edited 2D NMR and FT-ICR-MS, &multiplexed analysis by FT-ICR-MS;SA3. To develop web-based software for large-scale CS-adducted metabolite assignment &pathway reconstruction: Algorithm for robust automated MS assignment of metabolite &labeled isotopologues will be developed based on isotopologue cliques, FG profile, &molecular formula (MF). NMR- derived substructure &MF will be combined with MS data for semi-automated assignment of metabolite isotopomers &unknowns. Atom-resolved human metabolic database will be refined and tools developed for pathway reconstruction based on labeled metabolite profiles;SA4. To demonstrate the integrated CS profiling and biochemo-informatic approach in three basic and translational projects: Metabolic reprogramming driven by oncogenic gene defects or enzyme deletion in human lung and kidney cancers will be mapped using Stable Isotope-Resolved Metabolomics (SIRM) enhanced by newly developed CS tagging chemistry and automated assignment tools. Our long-term goal is to decipher human disease metabolic networks for drug discovery &early diagnosis.

Public Health Relevance

The proposed chemoselective tagging of metabolites with integrated informatics tools will enhance both metabolite profiling and elucidation of human disease metabolic networks far beyond current limits. The information gained from these tools will help accelerate at an unprecedented rate our functional understanding of human disease development and progression while, improving our ability to diagnose, prognose, and treat intractable human diseases such as cancer.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Special Emphasis Panel (ZRG1-BST-P (50))
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Balshaw, David M
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University of Louisville
Schools of Arts and Sciences
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Crooks, Daniel R; Maio, Nunziata; Lane, Andrew N et al. (2018) Acute loss of iron-sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells. J Biol Chem 293:8297-8311
Sun, Ramon C; Fan, Teresa W-M; Deng, Pan et al. (2017) Noninvasive liquid diet delivery of stable isotopes into mouse models for deep metabolic network tracing. Nat Commun 8:1646
Yang, Ye; Fan, Teresa W-M; Lane, Andrew N et al. (2017) Chloroformate derivatization for tracing the fate of Amino acids in cells and tissues by multiple stable isotope resolved metabolomics (mSIRM). Anal Chim Acta 976:63-73
Zhao, Jiangsha; Li, Jieran; Fan, Teresa W M et al. (2017) Glycolytic reprogramming through PCK2 regulates tumor initiation of prostate cancer cells. Oncotarget 8:83602-83618
Bruntz, Ronald C; Lane, Andrew N; Higashi, Richard M et al. (2017) Exploring cancer metabolism using stable isotope-resolved metabolomics (SIRM). J Biol Chem 292:11601-11609
Lane, Andrew N; Fan, Teresa W-M (2017) NMR-based Stable Isotope Resolved Metabolomics in systems biochemistry. Arch Biochem Biophys 628:123-131
Lane, Andrew N; Higashi, Richard M; Fan, Teresa W-M (2016) Preclinical models for interrogating drug action in human cancers using Stable Isotope Resolved Metabolomics (SIRM). Metabolomics 12:
Saxena, Neetu; Maio, Nunziata; Crooks, Daniel R et al. (2016) SDHB-Deficient Cancers: The Role of Mutations That Impair Iron Sulfur Cluster Delivery. J Natl Cancer Inst 108:
Li, Jing; Song, Jun; Zaytseva, Yekaterina Y et al. (2016) An obligatory role for neurotensin in high-fat-diet-induced obesity. Nature 533:411-5
Fan, Teresa W-M; Lane, Andrew N (2016) Applications of NMR spectroscopy to systems biochemistry. Prog Nucl Magn Reson Spectrosc 92-93:18-53

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