Метаболический Подтверждение пути и Discovery Через 13 C-маркировки протеиногенных Аминокислоты
Summary
13 С-изотопом маркировки полезным методом для определения ячейки центрального метаболизма для различных видов микроорганизмов. После клетки были культивировали с конкретными меченого субстрата, GC-MS измерение может выявить функциональные процессы обмена веществ на основе уникальных моделей маркировки в протеиногенных аминокислот.
Abstract
Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine cell central metabolism and discover new enzymes is 13C-assisted metabolism analysis 1. This technique is based on isotopic labeling, whereby microbes are fed with a 13C labeled substrates. By tracing the atom transition paths between metabolites in the biochemical network, we can determine functional pathways and discover new enzymes.
As a complementary method to transcriptomics and proteomics, approaches for isotopomer-assisted analysis of metabolic pathways contain three major steps 2. First, we grow cells with 13C labeled substrates. In this step, the composition of the medium and the selection of labeled substrates are two key factors. To avoid measurement noises from non-labeled carbon in nutrient supplements, a minimal medium with a sole carbon source is required. Further, the choice of a labeled substrate is based on how effectively it will elucidate the pathway being analyzed. Because novel enzymes often involve different reaction stereochemistry or intermediate products, in general, singly labeled carbon substrates are more informative for detection of novel pathways than uniformly labeled ones for detection of novel pathways3, 4. Second, we analyze amino acid labeling patterns using GC-MS. Amino acids are abundant in protein and thus can be obtained from biomass hydrolysis. Amino acids can be derivatized by N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) before GC separation. TBDMS derivatized amino acids can be fragmented by MS and result in different arrays of fragments. Based on the mass to charge (m/z) ratio of fragmented and unfragmented amino acids, we can deduce the possible labeled patterns of the central metabolites that are precursors of the amino acids. Third, we trace 13C carbon transitions in the proposed pathways and, based on the isotopomer data, confirm whether these pathways are active 2. Measurement of amino acids provides isotopic labeling information about eight crucial precursor metabolites in the central metabolism. These metabolic key nodes can reflect the functions of associated central pathways.
13C-assisted metabolism analysis via proteinogenic amino acids can be widely used for functional characterization of poorly-characterized microbial metabolism1. In this protocol, we will use Cyanothece 51142 as the model strain to demonstrate the use of labeled carbon substrates for discovering new enzymatic functions.
Protocol
Discussion
Этот протокол состоит из кормления ячейки с меченого субстрата и измерения в результате изотопного шаблонов маркировки в аминокислоты с помощью ГХ-МС. Поскольку MS данные (т / г соотношениях) дают только общее количество маркировки ионов MS, мы должны оценить isotopomer распределения аминоки?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
Это исследование было поддержано Карьера NSF Grant (MCB0954016) и биоэнергетики DOE исследовательских грантов (DEFG0208ER64694).
Materials
| Name of the reagent | Company | Catalogue number | Comments (optional) |
|---|---|---|---|
| TBDMS | Sigma-Aldrich | 19915 | – |
| THF | Sigma-Aldrich | 34865 | – |
| Labeled carbon substrate | Cambridge Isotope Laboratories | Depend on the experimental requirement | Website: http://www.isotope.com |
| Gas chromatograph | Agilent Technologies | Hewlett-Packard, model 7890A | – |
| GC Columns | J&W Scientific, Folsom, CA | DB5 (30m) | – |
| Mass spectrometer | Agilent Technologies | 5975C | – |
| Reacti-Vap Evaporator | Thermo Scientific | TS-18825 | For drying amino acid samples |
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