代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签
Summary
C – 13同位素标记是一个有用的技术，确定各类微生物细胞中央代谢。后已与一个特定的标记底物培养的细胞，气相色谱 – 质谱测量，可以揭示proteinogenic氨基酸的独特标签模式为基础的功能代谢途径。
Abstract
微生物可以利用生物化学和功能基因组学方法研究复杂的代谢途径。一个重要的技术，来研究细胞中央的新陈代谢，并发现新的酶是13的C -辅助代谢分析1。这种技术是基于同位素标记，用13 C标记底物的微生物美联储。通过跟踪在生化网络代谢物之间的原子过渡路径，我们可以判断功能的途径，并发现新的酶。
作为辅助方法，转录和蛋白质组学，isotopomer辅助代谢途径分析方法包含三个主要步骤： 首先 ，我们的成长与13 C标记底物的细胞。在这一步，中期和标记底物的选择组成的两个关键因素。为了避免从非标在营养补充剂的碳测量噪音与一个唯一碳源的培养基是必需的。此外，选择一个标记底物的基础上，如何有效地将澄清所分析的途径。由于新的酶往往涉及不同的反应的立体化学或中间产品，在一般情况下，单独标记的碳基板更新颖的途径检测信息统一标记的检测新颖的途径 3，4，第二，我们分析氨基酸的标签模式，采用GC – MS。氨基酸丰富的蛋白质，并，从而可以从生物质水解获得。氨基酸可GC分离之前的N -（叔丁基二甲基）- N – methyltrifluoroacetamide（TBDMS）衍生。 TBDMS衍生的氨基酸可分散MS和在不同的片段阵列。费（M / Z）零散和不分段氨基酸的比例群众的基础上，我们可以推断可能是中央的代谢产物的标记模式氨基酸的前体。第三，我们在建议的途径跟踪13C碳过渡，isotopomer数据的基础上，确认这些途径是否活跃2。氨基酸的测量提供了约8个中央代谢的关键前体代谢产物的同位素标记信息。这些代谢的关键节点，能反映相关中央通路的功能。
13的C协助通过proteinogenic氨基酸的代谢分析，可广泛用于低的特点微生物代谢功能特性。在这个协议中，我们将使用模型应变Cyanothece 51142证明标记的碳基板发现新酶功能的使用。
Protocol
1。细胞培养（图1） 生长在培养基细胞与微量元素，无机盐，维生素，并特别标明的碳基板是最佳途径调查。要么摇瓶或生物反应器细胞培养使用。有机营养物质，如酵母提取物，可能会干扰测量氨基酸的标签，因此不能在培养基中。 文化在一个最佳的波长（例如，外径730 Cyanothece 51142）的光密度与紫外/可见分光光度计监测细胞的生长。 可以先进行细胞生长?…
Discussion
该协议包括一个标记底物喂养的细胞，并测量通过GC – MS的氨基酸造成的同位素标记模式。由于MS数据（M / Z的比率），只是标签的MS离子的总金额，我们必须评估检查不分段（M – 57）+的m / z比值和零散的氨基酸的氨基酸isotopomer分布（即（M – 159）+（F302））。此外，我们可以执行几个细胞培养与化学相同的媒介，但有不同的标签模式（1 ST标记的位置，第二位置标示?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项研究是由美国国家科学基金会职业格兰特（MCB0954016）和能源部生物能源研究资助（DEFG0208ER64694）的支持。
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 |
Riferimenti
- Zamboni, N., Sauer, U. Novel biological insights through metabolomics and 13C-flux analysis. Curr. Opin. Microbiol. 12, 553-558 (2009).
- Tang, Y. J. Advances in analysis of microbial metabolic fluxes via 13C isotopic labeling. Mass. Spectrom. Rev. 28, 362-375 (2009).
- Tang, Y. J. Investigation of carbon metabolism in Dehalococcoides ethenogenes strain 195 via isotopic and transcriptomic analysisa. J. Bacteriol. 191, 5224-5231 (2009).
- Tang, Y. J. Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using GC-MS and FT-ICR mass spectrometry. Journal of Bacteriology. 189, 940-949 (2007).
- Dauner, M., Sauer, U. GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnology Progress. 16, 642-649 (2000).
- Wittmann, C. Fluxome analysis using GC-MS. Microbial Cell Factories. 6, 6-6 (2007).
- Antoniewicz, M. R., Kelleher, J. K., Stephanopoulos, G. Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis. Anal. Chem. 79, 7554-7559 (2007).
- Wahl, S. A., Dauner, M., Wiechert, W. New tools for mass isotopomer data evaluation in 13C flux analysis: mass isotope correction, data consistency checking, and precursor relationships. Biotechnology and Bioengineering. 85, 259-268 (2004).
- Shaikh, A., Tang, Y. J., Mukhopadhyay, A., Keasling, J. D. Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein. Analytical Chemistry. 80, 886-890 (2008).
- Tang, K. -. H., Feng, X., Tang, Y. J., Blankenship, R. E. Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114. PLoS One. 4, e7233-e7233 (2009).
- Tang, K. -. H. Carbon flow of Heliobacterium modesticaldum is more related to Firmicutes than to the green sulfur bacteria. J. Biol. Chem. 285, 35104-35112 (2010).
- Feng, X. Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. X514 via Isotopomer-Assisted Metabolite Analysis. Appl. Environ. Microbiol. 75, 5001-5008 (2009).
- Feng, X. Mixotrophic and photoheterotrophic metabolisms in Cyanothece sp. ATCC 51142 under continuous light. Microbiology. 156, 2566-2574 (2010).
- Tang, Y. J. Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-NTA. Appl. Environ. Microbiol. 73, 3859-3864 (2007).
- Tang, Y. J., Meadows, A. L., Kirby, J., Keasling, J. D. Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling. Journal of Bacteriology. 189, 894-901 (2007).
- Zhuang, W. Q. Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and the enhancement resulted to dechloronation activity. Appl. Environ. Microbiol. , (2011).
- Feng, X., Tang, K. -. H., Blankenship, R. E., Tang, Y. J. Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum. J. Biol. Chem. 285, 35104-35112 (2010).
- McKinlay, J. B., Harwood, C. S. Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proc. Natl. Acad. Sci. U.S.A. 107, (2010).
- McKinlay, J. B., Harwood, C. S. Calvin cycle flux, pathway constraints, and substrate oxidation state together determine the H2 biofuel yield in photoheterotrophic bacteria. MBio. 2, (2011).
- Erb, T. J. Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: The ethylmalonyl-CoA pathway. PNAS. 104, 10631-10636 (2007).
- Wu, B. Alternative isoleucine synthesis pathway in cyanobacterial species. Microbiology. 156, 596-602 (2010).
- Reddy, K. J., Haskell, J. B., Sherman, D. M., Sherman, L. A. Unicellular, aerobic nitrogen-ï¬xing cyanobacteria of the genus Cyanothece. J. Bacteriol. 175, 1284-1292 (1993).
- Shastri, A. A., Morgan, J. A. A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms. Phytochemistry. 68, 2302-2312 (2007).
- Tang, Y. J. Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbations. Biotechnol Prog. 25, 1254-1259 (2009).
- Zamboni, N., Fendt, S. M., Ruhl, M., Sauer, U. 13C-based metabolic flux analysis. Nature Protocols. 4, 878-892 (2009).
Tags
Metabolic Pathway ConfirmationMetabolic Pathway Discovery13C LabelingProteinogenic Amino AcidsBiochimicaFunctional Genomics13C-assisted Metabolism AnalysisIsotopic LabelingAtom Transition PathsBiochemical NetworkTranscriptomicsProteomicsIsotopomer-assisted Analysis Of Metabolic PathwaysLabeled SubstratesMinimal MediumCarbon SourceMeasurement NoisesNon-labeled CarbonNutrient SupplementsLabeled Substrate ChoiceReaction StereochemistryIntermediate ProductsSingly Labeled Carbon SubstratesUniformly Labeled SubstratesAmino Acid Labeling PatternsGC-MS
Citazione di questo articolo
You, L., Page, L., Feng, X., Berla, B., Pakrasi, H. B., Tang, Y. J. Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids. J. Vis. Exp. (59), e3583, doi:10.3791/3583 (2012).