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。

1。细胞培养（图1） <ol><li>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。 </li><li>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。 </li><li>可以先进行细胞生长在一个非标记的媒介。中间日志生长期细胞接种量接种率（3％（V / V））的标记介质​​的首选。标记的文化应该是在同一标记的介质，从最初的接种，以避免非标记的碳子培养（3％V / V接种比例）。 </li></ol><p类=“jove_title”&gt; 2。氨基酸提取<ol><li>收获子培养的细胞液（10ml）在中间通过离心（10分钟，8000 ×克）日志增长阶段。 </li><li> 6M盐酸1.5ml的悬浮颗粒，并转移到一个透明的玻璃，螺丝顶GC小瓶。第小瓶，放置24小时，他们在100 ° C烘箱生物量的蛋白质水解成氨基酸。生物质颗粒的水解可以产生20种常见氨基酸（图2）5 16 。半胱氨酸和色氨酸被降解，谷氨酰胺和天门冬酰胺，谷氨酸和天门冬氨酸，分别被转换。 </li><li>氨基酸溶液在20000 × g下离心5分钟用2毫升Eppendorf管，上清转移到新的GC小瓶。这一步水解液中删除的固体颗粒。 </li><li>卸下GC小瓶盖子和样品完全干下的空气流使用一个的Thermo Scientific Reacti - VAP蒸发器（注：冷冻干燥机，也可以采用tO）干样。这一步可以一蹴而就。 </li></ol> 3。氨基酸衍生GC - MS条件氨基酸或充电/高度极性的代谢产物通过气相色谱分析要求，这些代谢物的衍生物，使氨基酸波动，并可以由气相色谱 2分离。 <ol><li>溶于四氢呋喃（THF）与150μL和150μL的N -（叔丁基二甲基）N - methyltrifluoroacetamide衍生化试剂的干燥样品。 </li><li>所有样品° C为1小时65和80之间的烤箱或水浴孵育。涡偶尔确保样品瓶中的代谢产物被溶解。 </li><li>样品在20000 × g离心10分钟，然后将上清转移到新的GC小瓶。上清应该是一个明确的和黄的解决方案。由于探测器的饱和度，气相色谱 - 质谱测量精度可能会受到高concentration注入TBDMS衍生氨基酸（这些样品往往显示深褐色），因此，我们应该淡化这些样品使用四氢呋喃前的GC - MS测量6。 </li><li>分析样品的GC - MS（1:5或1:10的分光比，注射量= 1μL，载气为氦气= 1.2毫升/分钟）。使用下面的GC程序升温：保持在150 ° C 2分钟，增加3 ° C每分钟到280 ° C，增加在20 ° C每分钟300 ° C，然后按住5分钟。溶剂延迟可以设置为〜5分钟（30米GC柱）。质量电荷比（M / Z）在MS范围可设置在60和500之间。 </li></ol> 4。 GC - MS数据分析<ol><li> TBDMS衍生氨基酸的测量，也可以受GC分离同位素歧视。轻同位素移动速度稍快比GC柱升沉同位素。为了减少潜在的测量偏差，我们可能平均整个质谱氨基酸峰值范围6 </li><li>气相色谱和质谱TBDMS衍生代谢物已报告7日前。 GC保留时间，并为每个氨基酸独特的M / Z峰，如图3所示。 </li><li>氨基酸或中央代谢产物的衍生引入了大量包括自然标记同位素13℃（1.13％），18 O（0.20％），29 SI（4.70％），和30的Si（3.09 ％）。从原料质量isotopomer频谱的天然同位素的测量噪声可以修正通过发布的软件5， 8。最后的同位素标记数据报告质量分数，例如，M 0，M 1，M 2，M 3 和 M 4（代表包含零4个 13 C标记的碳的片段）。 </li><li>氨基酸的测定，可提供约8个关键的前体代谢物同位素标记信息：2 - 羰基- glutar吃了，3 - P -甘油，乙酰辅酶A，erythrose - 4 - P，草酰乙酸，磷酸，丙酮酸，和核糖- 5 - P。这些代谢产物的标签模式可以被用来确定几个中央代谢途径（图2）9。标记实验的结果可以得到进一步证实，用生物化学方法（如RT - PCR）。 </li></ol> 5。使用标记的氨基酸数据的途径分析通过调查只有少数关键从精心设计的13 C示踪实验制作的氨基酸，我们可以发现几个独特的途径或酶的活动，而无需执行复杂13的C代谢通量分析整个中央代谢。 <ol><li>恩特纳Doudoroff途径：[1 - 13 C]葡萄糖作为碳源。如果通路被激活，丝氨酸标签将明显比10丙氨酸标签。 </li><li>支TCA循环：[1 - 13 C]丙酮酸，可作为碳源。如果TCA循环被打破，天门冬氨酸，可以由两个碳原子标记，而谷氨酸是只有一个碳11，12标记。 </li><li> CO 2固定在混合营养代谢卡尔文-本森Bassham周期：非标记的CO 2和标有碳基板都被用作碳源。如果卡尔文循环功能，丝氨酸和组氨酸标签将被显著稀释，比其他氨基酸。这种方法可以相对确定的CO 2固定时，有机碳的来源，目前在中13。 </li><li>氧化磷酸戊糖途径：[1 - 13 C]葡萄糖作为碳源。如果通路被激活，非标丙氨酸将大于50％，12。 </li><li> Anaplerotic途径（例如，人教版+ CO 2 A草酰乙酸）：13 CO 2和非标记的碳基板（例如，甘油或PYRuvate）可以用来作为碳源。如果途径是积 ​​极的，天门冬氨酸标签将显著丰富，比较丙氨酸，丝氨酸13。 </li><li> 重新柠檬酸合酶：[1 - 13 C]丙酮酸，可作为碳源使用。如果激活酶，谷氨酸标记β-羧基3，4。 </li><li> Citramalate途径：[1 - 13 C]丙酮酸，[2 - 13 C]甘油，或[1 - 13彗星]醋酸可作为碳源。如果通路被激活，亮氨酸和异亮氨酸标签的金额是相同的 14 。 </li><li>丝氨酸-异柠檬酸裂合酶循环：[1 - 13 C]丙酮酸或[1 - 13彗星]乳酸可以作为碳源。如果通路被激活，在第三的位置碳丝氨酸将被标记为15。 </li><li>利用营养素（即外源性氨基酸）：一个完全标记的碳基板和非标记的氨基酸的培养基可以使用。如果细胞选择性地利用这些非标补充养分，我们将看到这些氨基酸在生物量的显著标签稀释。这种方法可以用来研究细胞16首选营养补充剂。 </li></ol> 6。代表性的成果最近生物能源的研究，在使用新的光合微生物的生物能源生产恢复的利益和CO 2捕获。在过去的几年中，不少13C -代谢分析，包括先进的13C代谢通量分析（13C - MFA），已被应用于调查中央光合细菌的代谢，因为中央的代谢途径的生化知识是没有依据在这些非模型生物体10，11，17-20。在这里，我们目前的一个备用异亮氨酸途径Cyanothece发现51142 21的例子。青色othece 51142不含有酶（EC 4.3.1.19，苏氨酸氨酶），苏氨酸转化为2 - ketobutyrate催化典型的异亮氨酸合成途径。要解决的异亮氨酸的途径，我们成长在ASP2 中等 22 Cyanothece 51142（20毫升）与甘油（2 - 13C，&gt; 98％）54毫米。Cyanothece 51142 采用了第二的位置标示为主要碳源的甘油。我们观察到，苏氨酸和丙氨酸有一个标记的碳，而异亮氨酸是与三个碳原子标记。因此，在Cyanothece 51142合成不能被来自受聘于大多数生物体（图4）中苏氨酸路线。另一方面，亮氨酸和异亮氨酸具有相同标签的基础上的片段模式（M - 15）+片段（M - 159）+ 。例如，从isotopomer数据[M - 15] +（含不分段氨基酸）相同的标签显示为亮氨酸（M0 = 0.01，货币供应量M1 = 0.03，M2 = 0.21，M3 = 00.69）和异亮氨酸（M0 = 0.01，M1 = 0.03，M2 = 0.24，M3 = 0.67）。因此，必须从相同的前体（即丙酮酸和乙酰辅酶A）合成亮氨酸和异亮氨酸。这个观察citramalate异亮氨酸合成途径中的标有“低碳转型是一致的。为了证实这一途径，我们搜索的联合基因组研究所数据库，并找到一个citramalate 合酶 CIMA（cce_0248） 在 Cyanothece存在。 <img src="/files/ftp_upload/3583/3583fig1.jpg" alt="图1"/> 图1 13 C辅助途径分析步骤。 <img src="/files/ftp_upload/3583/3583fig2.jpg" alt="图2"/> 图2用于收购其代谢的前体的标签模式的氨基酸。 ACOA，乙酰辅酶A; AKG，α-酮戊二酸; C5P，核糖-5 - 磷酸;企业所得税，柠檬酸; E4P，erythrose 4 - 磷酸; G6P，葡萄糖-6 - 磷酸;高龄津贴，<html>草酰乙酸;人教版，磷酸;美巡赛，3 - 磷酸; PYR，丙酮酸。 <img src="/files/ftp_upload/3583/3583fig3.jpg" alt="图3"/> 图3。气相色谱峰为16个氨基酸。 TBDMS衍生氨基酸破获两个片段：由MS（M - 57），包含整个氨基酸，和（M - 159）+，缺乏α氨基酸的羧基。亮氨酸和异亮氨酸，（M - 57）+其他的质谱峰重叠。我们建议使用片段（M - 15）+分析整个氨基酸标签。 （F302）+组进行检测，大多数氨基酸，其中包含仅在第（α-羧基组）和氨基酸骨干的第二个碳原子。因为这ms峰值往往具有较高的噪声信号的比值，（F302）+不建议 7代谢通量定量分析。 igure 4“/&gt; 图4。异亮氨酸途径Cyanothece 51142（从我们以前的文件修改）21标签转换。

<ol>
	<li>Zamboni, N., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+biological+insights+through+metabolomics+and+13C-flux+analysis.">Novel biological insights through metabolomics and 13C-flux analysis.</a> Curr. Opin. Microbiol. 12, 553-558 (2009).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+analysis+of+microbial+metabolic+fluxes+via+13C+isotopic+labeling.">Advances in analysis of microbial metabolic fluxes via 13C isotopic labeling.</a> Mass. Spectrom. Rev. 28, 362-375 (2009).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+carbon+metabolism+in+Dehalococcoides+ethenogenes+strain+195+via+isotopic+and+transcriptomic+analysisa.">Investigation of carbon metabolism in Dehalococcoides ethenogenes strain 195 via isotopic and transcriptomic analysisa.</a> J. Bacteriol. 191, 5224-5231 (2009).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathway+confirmation+and+flux+analysis+of+central+metabolic+pathways+in+Desulfovibrio+vulgaris+Hildenborough+using+GC-MS+and+FT-ICR+mass+spectrometry.">Pathway confirmation and flux analysis of central metabolic pathways in Desulfovibrio vulgaris Hildenborough using GC-MS and FT-ICR mass spectrometry.</a> Journal of Bacteriology. 189, 940-949 (2007).</li><li>Dauner, M., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GC-MS+analysis+of+amino+acids+rapidly+provides+rich+information+for+isotopomer+balancing.">GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing.</a> Biotechnology Progress. 16, 642-649 (2000).</li><li>Wittmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluxome+analysis+using+GC-MS.">Fluxome analysis using GC-MS.</a> Microbial Cell Factories. 6, 6-6 (2007).</li><li>Antoniewicz, M. R., Kelleher, J. K., Stephanopoulos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+assessment+of+amino+acid+mass+isotopomer+distributions+for+metabolic+flux+analysis.">Accurate assessment of amino acid mass isotopomer distributions for metabolic flux analysis.</a> Anal. Chem. 79, 7554-7559 (2007).</li><li>Wahl, S. A., Dauner, M., Wiechert, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+tools+for+mass+isotopomer+data+evaluation+in+13C+flux+analysis:+mass+isotope+correction,+data+consistency+checking,+and+precursor+relationships.">New tools for mass isotopomer data evaluation in 13C flux analysis: mass isotope correction, data consistency checking, and precursor relationships.</a> Biotechnology and Bioengineering. 85, 259-268 (2004).</li><li>Shaikh, A., Tang, Y. J., Mukhopadhyay, A., Keasling, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotopomer+distributions+in+amino+acids+from+a+highly+expressed+protein+as+a+proxy+for+those+from+total+protein.">Isotopomer distributions in amino acids from a highly expressed protein as a proxy for those from total protein.</a> Analytical Chemistry. 80, 886-890 (2008).</li><li>Tang, K. -. H., Feng, X., Tang, Y. J., Blankenship, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+metabolism+and+carbon+fixation+in+Roseobacter+denitrificans+OCh114.">Carbohydrate metabolism and carbon fixation in Roseobacter denitrificans OCh114.</a> PLoS One. 4, e7233-e7233 (2009).</li><li>Tang, K. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+flow+of+Heliobacterium+modesticaldum+is+more+related+to+Firmicutes+than+to+the+green+sulfur+bacteria.">Carbon flow of Heliobacterium modesticaldum is more related to Firmicutes than to the green sulfur bacteria.</a> J. Biol. Chem. 285, 35104-35112 (2010).</li><li>Feng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+Central+Metabolic+Pathways+in+Thermoanaerobacter+sp.+X514+via+Isotopomer-Assisted+Metabolite+Analysis.">Characterization of the Central Metabolic Pathways in Thermoanaerobacter sp. X514 via Isotopomer-Assisted Metabolite Analysis.</a> Appl. Environ. Microbiol. 75, 5001-5008 (2009).</li><li>Feng, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mixotrophic+and+photoheterotrophic+metabolisms+in+Cyanothece+sp.+ATCC+51142+under+continuous+light.">Mixotrophic and photoheterotrophic metabolisms in Cyanothece sp. ATCC 51142 under continuous light.</a> Microbiology. 156, 2566-2574 (2010).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flux+analysis+of+central+metabolic+pathways+in+Geobacter+metallireducens+during+reduction+of+soluble+Fe(III)-NTA.">Flux analysis of central metabolic pathways in Geobacter metallireducens during reduction of soluble Fe(III)-NTA.</a> Appl. Environ. Microbiol. 73, 3859-3864 (2007).</li><li>Tang, Y. J., Meadows, A. L., Kirby, J., Keasling, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anaerobic+central+metabolic+pathways+in+Shewanella+oneidensis+MR-1+reinterpreted+in+the+light+of+isotopic+metabolite+labeling.">Anaerobic central metabolic pathways in Shewanella oneidensis MR-1 reinterpreted in the light of isotopic metabolite labeling.</a> Journal of Bacteriology. 189, 894-901 (2007).</li><li>Zhuang, W. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+utilization+of+exogenous+amino+acids+by+Dehalococcoides+ethenogenes+strain+195+and+the+enhancement+resulted+to+dechloronation+activity.">Selective utilization of exogenous amino acids by Dehalococcoides ethenogenes strain 195 and the enhancement resulted to dechloronation activity.</a> Appl. Environ. Microbiol. , (2011).</li><li>Feng, X., Tang, K. -. H., Blankenship, R. E., Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+flux+analysis+of+the+mixotrophic+metabolisms+in+the+green+sulfur+bacterium+Chlorobaculum+tepidum.">Metabolic flux analysis of the mixotrophic metabolisms in the green sulfur bacterium Chlorobaculum tepidum.</a> J. Biol. Chem. 285, 35104-35112 (2010).</li><li>McKinlay, J. B., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbon+dioxide+fixation+as+a+central+redox+cofactor+recycling+mechanism+in+bacteria.">Carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria.</a> Proc. Natl. Acad. Sci. U.S.A. 107, (2010).</li><li>McKinlay, J. B., Harwood, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calvin+cycle+flux,+pathway+constraints,+and+substrate+oxidation+state+together+determine+the+H2+biofuel+yield+in+photoheterotrophic+bacteria.">Calvin cycle flux, pathway constraints, and substrate oxidation state together determine the H2 biofuel yield in photoheterotrophic bacteria.</a> MBio. 2, (2011).</li><li>Erb, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+C5-dicarboxylic+acids+from+C2-units+involving+crotonyl-CoA+carboxylase/reductase:+The+ethylmalonyl-CoA+pathway.">Synthesis of C5-dicarboxylic acids from C2-units involving crotonyl-CoA carboxylase/reductase: The ethylmalonyl-CoA pathway.</a> PNAS. 104, 10631-10636 (2007).</li><li>Wu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+isoleucine+synthesis+pathway+in+cyanobacterial+species.">Alternative isoleucine synthesis pathway in cyanobacterial species.</a> Microbiology. 156, 596-602 (2010).</li><li>Reddy, K. J., Haskell, J. B., Sherman, D. M., Sherman, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unicellular,+aerobic+nitrogen-ï¬xing+cyanobacteria+of+the+genus+Cyanothece.">Unicellular, aerobic nitrogen-ï¬xing cyanobacteria of the genus Cyanothece.</a> J. Bacteriol. 175, 1284-1292 (1993).</li><li>Shastri, A. A., Morgan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transient+isotopic+labeling+methodology+for+13C+metabolic+flux+analysis+of+photoautotrophic+microorganisms.">A transient isotopic labeling methodology for 13C metabolic flux analysis of photoautotrophic microorganisms.</a> Phytochemistry. 68, 2302-2312 (2007).</li><li>Tang, Y. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invariability+of+central+metabolic+flux+distribution+in+Shewanella+oneidensis+MR-1+under+environmental+or+genetic+perturbations.">Invariability of central metabolic flux distribution in Shewanella oneidensis MR-1 under environmental or genetic perturbations.</a> Biotechnol Prog. 25, 1254-1259 (2009).</li><li>Zamboni, N., Fendt, S. M., Ruhl, M., Sauer, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=13C-based+metabolic+flux+analysis.">13C-based metabolic flux analysis.</a> Nature Protocols. 4, 878-892 (2009).</li></ol>

没有利益冲突的声明。

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?...

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td> TBDMS </td><td> Sigma - Aldrich公司</td><td> 19915 </td><td> - </td></tr><tr><td>四氢呋喃</td><td> Sigma - Aldrich公司</td><td> 34865 </td><td> - </td></tr><tr><td>标有“碳源</td><td>剑桥同位素实验室</td><td>依赖对实验的要求</td><td>网址： <a href="http://www.isotope.com">http://www.isotope.com</a> </td></tr><tr><td>气相色谱仪</td><td>安捷伦科技公司</td><td>惠普，型号7890A </td><td> - </td></tr><tr><td> 气相色谱柱 </td><td> J＆W科学，福尔瑟姆，CA </td><td> DB5（30米） </td><td> - </td></tr><tr><td>质谱仪</td><td>安捷伦科技公司</td><td> 5975C </td><td> - </td></tr><tr><td> Reacti - VAP Evaporator </td><td> Thermo Scientific的</td><td> TS - 18825 </td><td>对于干燥氨基酸样品</td></tr></tbody></table>

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

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。

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

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。

代谢途径的确认和发现通过 13 C - Proteinogenic氨基酸的标签

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

metabolic-pathway-confirmation-discovery-through-13c-labeling

Research

JoVE Journal

Biology

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

24.1K 次观看.  Washington University. 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。

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

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

液相恢复GELFREE 8100分馏系统使用基于分子量分馏

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

科研

生物学

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

一步代谢：碳水化合物，在一个单一的过程量化的有机和氨基酸

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop微量核酸的定量

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

斑马鱼胚胎的代谢谱分析

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

代谢率的测量<em&gt;果蝇</em&gt;使用呼吸运动

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

的蛋白质 - 蛋白质相互作用抑制剂的发展，通过替换：应用程序的设计和开发非ATP竞争性CDK抑制剂

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

非经典氨基酸的具体残留纳入到模型蛋白质使用<em&gt;大肠杆菌</em&gt;无细胞转录翻译系统

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤：一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.