Name: metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids
Uploaded: 2012-01-26T13:00:00
Description: Watch this Scientific Journal Video about Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids at JoVE.com

This study was supported by an NSF Career Grant (MCB0954016) and a DOE Bioenergy Research Grant (DEFG0208ER64694).

1.	Cell culture (Figure 1)
<ol>
	<li>Grow cells in minimal medium with trace elements, salts, vitamins, and specifically labeled carbon substrates that are best for pathway investigation. Use either shaking flasks or bioreactors for cell culture. Organic nutrients, such as yeast extract, may interfere with the measurement of amino acid labeling and thus cannot be present in the culture medium.	</li>
	<li>Monitor cell growth by the optical density of the culture at an optimal wavelength (e.g., OD730 for...

<ol>
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This protocol consists of feeding the cell with a labeled substrate and measuring the resulting isotopic labeling patterns in the amino acids via GC-MS. Since MS data (m/z ratios) give just the overall amount of labeling of MS ions, we have to assess the isotopomer distributions of amino acids by examining the m/z ratios of both unfragmented (M-57)+ and fragmented amino acids (i.e., (M-159)+ and (f302)+). Furthermore, we can perform several cell cultures with a chemically identical medium...

<table border="1">
<tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 <td>TBDMS</td>
 <td>Sigma-Aldrich</td>
 <td>19915</td>
 <td>-</td>
 </tr>
 <tr>
 <td>THF</td>
 <td>Sigma-Aldrich</td>
 <td>34865</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Labeled carbon substrate</td>
 <td>Cambridge Isotope Laboratories</td>
 <td>Depend on the experimental requirement</td>
 <td>Website:
 <a href="http://www.isotope.com">http://www.isotope.com</a></td>
 </tr>
 <tr>
 <td>Gas chromatograph</td>
 <td>Agilent Technologies</td>
 <td>Hewlett-Packard, model 7890A</td>
 <td>-</td>
 </tr>
 <tr>
 <td>GC Columns</td>
 <td>J&amp;W Scientific, Folsom, CA</td>
 <td>DB5 (30m)</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Mass spectrometer</td>
 <td>Agilent Technologies</td>
 <td>5975C</td>
 <td>-</td>
 </tr>
 <tr>
 <td>Reacti-Vap Evaporator</td>
 <td>Thermo Scientific</td>
 <td>TS-18825</td>
 <td>For drying amino acid samples</td>
 </tr>
</tbody>
</table>

metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids

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.

Watch this Scientific Journal Video about Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids at JoVE.com

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13C-isotope labeling is a useful technique for determining the cell central metabolism for various types of microorganisms. After cells have been cultured with a specific labeled substrate, GC-MS measurement can reveal functional metabolic pathways based on unique labeling patterns in proteinogenic amino acids.

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

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