The authors would like to thank Dr. Anas Kamleh for valuable help with optimizing mass spectrometry methods, and Annika von Vollenhoven for assistance with cell sorting. This research was supported by the Swedish Foundation for Strategic Research (grant no. FFL12-0220) and the Strategic Programme in Cancer Research (IR, RN); the Swedish Heart-Lung Foundation (CEW, HG); and Mary Kay Foundation (JW, MJ).

1.提取代谢物<ol><li> 从盘中提取 <ol><li>在稳定同位素标记的培养基+透析补充剂（血清或其他生长补充剂）一式三份一6孔板培养细胞直至细胞成为75％汇合。 注意:在此培养HeLa细胞在RPMI 48小时含有40％U- 13 C-葡萄糖和70％U- 13 C，15 N 2 -谷氨酰胺和5％透析的FBS（胎牛血清）。透析FBS用来摆脱可能污染标记的媒体小分子量代谢产物。培养在之前的实际实验透析补充细胞建议确保细胞在培养基中正常生长。补充剂在0.15M NaCl的溶液透析使用蛇皮透析管过夜。 </li><li>在萃取丢弃培养基的当天，漂洗孔两次用500微升冷的HBSS，然后丢弃它。 注意:这里使用含40％HBSS U-13 C-葡萄糖，因为它也是在培养基中。 </li><li>添加600μL的100％甲醇中预冷干冰上。 </li><li>传送盘干冰和除去细胞材料用细胞刮刀。 </li><li>仔细吸取细胞提取物到微量离心管中并储存在-80℃直至质谱分析。 </li></ol></li><li> 模拟排序细胞的提取 <ol><li>培养细胞在100毫米的菜一式三份的稳定同位素标记的培养基+透析补充剂。 注:这里的文化HeLa细胞在100mm皿48小时，因为（〜4×10 6）需要的细胞大量获得500000排序细胞。被要求HeLa细胞提取物这个数目，以获得代谢产物的良好的测量。所用的培养基是RPMI含40％U- 13 C葡萄糖和70％U- 13 C，15 N2谷氨酰胺和5％FBS透析。 </li><li>在萃取后，弃去培养基的一天，冲洗井瓦特第i个1.5毫升温HBSS，然后丢弃它。 </li><li>通过在37℃下加入1.5毫升胰蛋白酶/ EDTA 4分钟分离细胞。执行在4℃或在冰以下步骤。 </li><li>通过加入3毫升冰冷的HBSS（Hank氏平衡盐溶液）+透析补充停用胰蛋白酶。 </li><li>收集在15毫升管中并离心细胞，在750×g离心3分钟。 </li><li>悬浮在HBSS +透析补充+ 1mM EDTA中沉淀的浓度1-2×10 6个细胞/ mL，通过40微米的细胞滤网，以获得单细胞，并转移到一个5毫升管中。 </li><li>仅用于汗衫排序细胞通过细胞分选仪门控，并在750×g离心在4℃下3分钟的离心分选的细胞。 注:此处的排序HeLa细胞的速率1,000个事件/秒，用仪表压力27磅，并用100微米的喷嘴。 HeLa细胞是大所以他们应该在一个缓慢的事件发生率和大喷嘴进行排序，以获得尽可能完整的颗粒越好。保持所述细胞在冷块throughouŧ排序，以降低新陈代谢。排序过程的全面描述以前12中所述。 </li><li>弃上清，悬浮颗粒在50微升冰冷的dh 2 O加入甲醇之前获得均匀的颗粒。冷甲醇除了直接沉淀形成固体沉淀，这是很难再悬浮。 </li><li>通过加入保持在干冰540微升甲醇提取代谢物，并在-80保持萃取℃直至液相色谱 - 高分辨质谱（LC-HRMS）分析。 </li></ol></li><li> 细胞周期的提取分选的细胞 <ol><li>培养细胞在文化传媒+透析一式三份补充100毫米菜。 注:含有Geminin Fucci绿色（MAG1-hGem）探测器13，它允许为G1（负）和SG2M（阳性）细胞的分选这里培养HeLa细胞。细胞可在一个稳定的同位素示踪培养基中培养悠久的要求。在这里，我们培养他们在未标记的媒体46小时，然后我们切换到含40％U- 13 C葡萄糖和70％U- 13 C RPMI，前15 N2谷氨酰胺2小时开始排序。这是为了能够研究这需要一个短脉冲标记细胞周期阶段完成。 </li><li>在萃取后，弃去培养基的一天，冲洗用1.5ml温暖的HBSS孔中，然后将其丢弃。 </li><li>通过在37℃下加入1.5毫升胰蛋白酶/ EDTA 4分钟分离细胞。执行在4℃或在冰以下步骤。 </li><li>通过加入3毫升冰冷的HBSS +透析的补充停用胰蛋白酶。 </li><li>收集在15毫升管中并离心细胞，在750×g离心3分钟。 </li><li>悬浮在HBSS +透析补充+ 1mM EDTA中沉淀的浓度1-2×10 6个细胞/ mL，通过40微米的细胞滤网，以获得单细胞，并转移到一个5毫升管中。 </li><li>排序细胞通过细胞分选仪克阿婷出碎屑和双峰，然后选通为感兴趣的细胞标记物，并离心分选的细胞在750×g离心在4℃下3分钟。 注:此处的排序HeLa细胞的速率1,000个事件/秒，用仪表压力27磅，并用100微米的喷嘴。保持在冷块中的细胞在整个排序以降低代谢速率。 </li><li>弃上清，悬浮颗粒在50微升冰冷的dh 2 O加入甲醇之前获得均匀的颗粒。冷甲醇除了直接沉淀形成固体沉淀，这是很难再悬浮。 </li><li>通过加入保持在干冰540微升甲醇提取代谢物，并在-80℃保持提取直到LC-HRMS的分析。 </li></ol></li></ol> 2.质谱分析注:在这里，我们描述了协议的LC-HRMS系统上分析细胞提取物。可用于细胞提取物的分析的任何代谢的方法。全扫描分析可能是用于检测广泛的代谢物是有用的。 <ol><li>校准用质谱参考校准混合仪器。 </li><li>在冰上解冻细胞提取物30分钟，并涡旋15秒。 </li><li>以13,000 xg离心转移100μL的细胞提取物与一个旋转过滤器，离心10分钟，在4℃。 </li><li>注入滤液12.5微升到在LC-HRMS的系统。 </li><li>使用两性离子亲水相互作用液相色谱（ZIC-HILIC）柱（150毫米×4.6毫米，5微米颗粒尺寸）分离代谢物嵌合用0.1％甲酸的梯度洗脱一个ZIC-HILIC保护柱英寸（20毫米×2.1 MM）酸在水（溶剂A）和乙腈（溶剂B）。在溶剂A为20％开始梯度洗脱，并在17分钟增加至80％。保持在23℃和400微升的流分-1和柱温和样品盘4℃在4分钟该百分比，分别。 </li><li>使用Instrument耦合到用于代谢物的检测，在正的和负的模式作为电离源加热的电（H-ESI II）中，并在70000全宽度半最大值（FWHM）的质量分辨能力完全扫描采集模式色谱分离（ M / Z 200）。 </li><li>用于鞘气和辅助气体氮气（纯度&gt; 99.995％）以45的流速和10 AU（任意单位），并设置蒸发器温度为350℃，并在正模式和-3.5千伏4千伏的电电压在否定模式。 </li></ol> 3.数据分析<ol><li>选择一些代谢物的量的标准是可用的并且其显示样品中良好的质量峰。良好的质量峰具有高信号噪声比。它验证峰值质量，确保不包含虚假同位素是很重要的。这在形状和/或保留时间不同同位素峰可能假。 </li><li>对于同位素标记的样本计算的质量同位素（MI）通过将每心肌梗死的峰面积与所有多边的总峰面积分数。 </li><li>分别计算标记碳/氮分数，13 C和15 N，聚财，为 <img alt="式（1）" src="/files/ftp_upload/55011/55011eq1.jpg" /> 其中n是在代谢物（分别或氮，）的总碳数，混匀是x的MI分数。 注:所有计算可以使用的编程语言来执行。 </li></ol>

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作者什么都没有透露。

我们的方法是基于这样的原则，在细胞代谢产物的MID反映的细胞代谢活动的"历史"。这使得有可能研究的代谢活性在细胞的亚群，因为它们发生在细胞的复杂社区，细胞分选过程之前。与此相反，代谢物的峰面积分选的细胞，并从培养皿11直接提取的提取物之间明显不同。这部分是因为不同的化学组成改变在质谱，一个所谓的"基体效应"的信号响应，但是我们还表明，氨基酸选自细胞分选过程中丢失，而细胞被保持在缓冲器11。这可以在排序过程中改变的代谢状态和细胞的活动，但是这是在本身无关我们的方法:提供该MID的是合理地接近于在直接提取观察到的，数据仍然瓦利在原有的复杂的文化各亚群的代谢状态的三维测量。 我们最初测试，其中细胞分类直接进入提取液（甲醇）迅速淬火新陈代谢的方法。不幸的是，所得到的提取物难以分析，因为它们含有大量的盐和可能的其它污染物从细胞分选仪鞘液，导致我们的质谱系统大规模离子抑制。因此，我们定居在这里所描述的协议，其中由细胞分选仪沉积过多流体萃取之前冲了出去。 我们的协议嗣继承排序在HBSS，补充有葡萄糖，以保持细胞生存力生理盐溶液。在某些方面，可以优选进行排序的细胞中的实际培养基代谢应激最小化如氨基酸泄漏。然而，这是很难洗分选的细胞的小球团，并theref目前在矿石中的代谢物会污染胞内代谢产物的追捧移动互联网设备。每当13 C标记的葡萄糖被用作示踪剂，相同的标记的葡萄糖应在的HBSS溶液中使用为好。 如在图2中看到的，许多，但不是所有的代谢物，所述细胞分拣过程后保持它们的MID。我们不知道为什么某些代谢产物（乳酸，例如）是专门改变。这一结果强调，关键是要验证的移动互联网设备感兴趣的是对由细胞分选模拟程序进行排序强劲。虽然这是不可能直接验证排序亚群的MID，MID的是由模拟排序（谷氨酸，例如）的影响应该是由实际的分拣，以及，作为程序是除了小区选择相同的影响。此验证步骤应进行每当使用新的细胞类型或培养条件。它指出，重要的是Ò乌尔方法局限于为哪些健壮的MID可以以这种方式来验证代谢物。 我们预计，在这里所描述的方法将在若干细胞生物学和生物医学中的应用是有用的。实例包括共培养细胞的代谢表型，例如造血干细胞-饲养层培养物，神经元星形胶质细胞模型4，血细胞2的亚群，并且还从动物模型分离复杂的细胞群。

高等生物包含协作带来更复杂的功能不同的细胞类型的复杂的社区。例如，肿瘤不仅含有癌细胞，而且成纤维细胞，在构成血管，并经常免疫细胞浸润1细胞;血液中含有数十免疫细胞亚型2的复杂混合物;甚至培养的细胞系可以由多个亚群，如管腔和乳腺癌细胞3的基底亚型。此外，不同的细胞类型共存能够表现出代谢"合作"。例如，在大脑中，星形胶质细胞被认为是葡萄糖转化为乳酸，然后将其"喂"到氧化该衬底4的神经元; T淋巴细胞是在一些依赖于相邻的树突细胞作为半胱氨酸5的源上下文;和癌细胞可能与阿索协作肿瘤6 ciated成纤维细胞。为了理解这种系统的代谢行为，至关重要的是，以分离和测量存在于各种细胞类型的代谢活动。 迄今为止用于分离细胞类型的最常用的方法是荧光激活细胞分选。此方法是广泛适用的，只要该细胞类型或感兴趣的状态可以"标记"使用荧光抗体，工程化的荧光蛋白的表达，或其他的染料。一种选择是最初分离的细胞类型通过细胞分选仪，再培养得到的个别细胞类型，然后执行这些培养7的代谢研究。然而，如果细胞类型或表型是在培养条件下稳定的，并且不能捕获瞬态行为，如细胞周期状态，也不会在共培养物的代谢合作，这是唯一可行的。对于这样的情况下，代谢必须直接上，以便测量rted细胞。这是具有挑战性的，因为细胞分选过程科目细胞可能扭曲他们的新陈代谢8的压力，而我们都知道只有少数的研究采用这种方法9,10。特别是，我们已经发现，主要代谢产物如氨基酸可以从保持在细胞分拣缓冲液细胞泄漏，使绝对代谢物丰度的测量结果不再可靠11（虽然排序级分之间的相对比较，仍可能是有价值的）。 为了规避这些问题，我们的标签细胞之前，分拣稳定同位素，并专注于细胞代谢产物，而不是代谢产物丰度的MID产品。由于MID产品则经过较长时间尺度上形成的，就应该少受短期接触到排序的条件。我们用量化的全扫描高分辨率质谱，这是非常敏感地提供移动互联网设备达TA数以百计的代谢物从约50选细胞出发，大约需要30-60分钟的细胞分选的时间。一个之间的比较"模拟排序"控制（通过细胞分选仪仪器没有选通任何特定人口传递细胞）和代谢物提取直接从培养皿制成，以确保所观察到的MID的是代表那些在原始培养物。取决于稳定同位素示踪剂的选择，各种代谢途径可以用这种方法进行研究。

<table><tbody><tr><td>HBSS</td><td>Sigma</td><td>H6648</td><td></td></tr><tr><td>INFLUX (inFlux v7 Sorter)</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>U-&lt;sup&gt;13&lt;/sup&gt;C-Glucose</td><td>Cambridge isotopes</td><td>40762-22-9 / GLC-018</td><td></td></tr><tr><td>U-&lt;sup&gt;13&lt;/sup&gt;C,&lt;sup&gt;15&lt;/sup&gt;N2-Glutamine</td><td>Cambridge isotopes</td><td>CNLM-1275-H-0.1</td><td></td></tr><tr><td>Methanol (JT Baker), HPLC grade</td><td>VWR</td><td>BAKR8402.2500</td><td></td></tr><tr><td>Ultrafree - MC - VV centrifugal Filters. Durapore PVDF 0.1 &amp;micro;m</td><td>Millipore</td><td>UFC30VV00</td><td></td></tr><tr><td>Ultimate 3,000 UHPLC</td><td>Thermo Fisher scientific</td><td></td><td></td></tr><tr><td>Q-Exactive Orbitrap Mass spectrometer</td><td>Thermo Fisher scientific</td><td></td><td></td></tr><tr><td>Merk-Sequant ZIC HILIC column (150 mm x 4.6&amp;nbsp;mm, 5 &amp;micro;m)</td><td>Merck KGaA</td><td>1.50444.0001</td><td></td></tr><tr><td>Merk-Sequant ZIC HILIC guard column (20 mm x 2.1 mm)</td><td>Merck KGaA</td><td></td><td></td></tr><tr><td>Acetonitrile Optima LC-MS, amber glass</td><td>Fisher Scientific</td><td>A955-212</td><td></td></tr><tr><td>Milli-Q water</td><td>Millipore</td><td></td><td>Produced with a Milli-Q Gradient system</td></tr><tr><td>Myrsyra 99.5% Optima (Formic acid)</td><td>Fisher Scientific</td><td>11423423</td><td></td></tr><tr><td>X100 Screw Vial 1.5 ml, 8-425 32x11.6 mm, amber, 100 units</td><td>Thermo Fisher scientific</td><td>10560053</td><td></td></tr><tr><td>X100 Lock Skruv Vitt PTFE Packing 8-425 (Screw caps)</td><td>Thermo Fisher scientific</td><td>12458636</td><td></td></tr><tr><td>ProteoMass LTQ/FT-Hybrid ESI Pos. Mode Cal Mix</td><td>Sigma-Aldrich</td><td>MSCAL5</td><td>Calibration kit</td></tr><tr><td>SNAKESKIN 10K MWCO&amp;nbsp;</td><td>Thermo Fisher scientific</td><td>88245</td><td></td></tr><tr><td>Mathematica v.10&amp;nbsp;</td><td>Wolfram Research</td><td></td><td></td></tr></tbody></table>

a method for measuring metabolism in sorted subpopulations of complex cell communities using stable isotope tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

作为一个例子，在这里，我们描述了一种实验调查HeLa细胞根据细胞周期相排序的代谢。要标记范围广两个碳原子和氮代谢的中心，我们培养的细胞用U- 13 C葡萄糖和U- 13 C，15 N谷氨酰胺作为示踪剂48小时。要获取验证实验丰富移动互联网设备，我们在这里选择了40％的U- 13 C葡萄糖和70％U- 13 C，15 N2谷氨酰胺混合后作为同位素的中等水平往往产生更多样化的MI模式14。 为了验证实验中，我们进行了85代谢产物有针对性的分析。质量控制步骤以删除质量较差LC-MS的峰后，我们能够探测到在直接细胞提取物，其中66存在于模拟分选的细胞（96％）69的峰。其中60出现了标记（同位素富集上述自然丰度），和在大多数情况下，它们的MID是菜提取物和模拟分选的细胞（ 图2A）之间的相似。例如，谷氨酸的MID（其中包括13 C和15 N MIS）是盘和模拟排序萃取（ 图2B）之间的相似，表明谷氨酸MID是在分选的细胞群体也可靠。然而，一些代谢产物显然受排序过程:例如，乳酸盐在模拟选的细胞过少13 的 C 3，原因不明。这种代谢产物应谨慎分析从实际分数排序数据时进行查看。在模拟分选的细胞，测得的MI分数为91％的标准偏差小于跨越生物三份1％，表明MID的是在分选的细胞高度重现。 接下来，我们分析HeLa细胞分成两种G0 / G1或S / G2 / M细胞周期小号使用FUCCI Geminin探头12每日新闻。由于这些细胞周期阶段仅持续约10小时，我们在这里"脉冲"标记为2小时培养，实现阶段之间的不同同位素标记。例如，我们注意到，胞苷标记在两个群体，但在S期在S / G2 / M阶段更高富集，具有增加的从头核苷酸合成是一致的（ 图3）。在这种情况下，在MID显示在这两个级分相同的图案，这表明相同的合成途径被使用，但是更积极的在S / G2 / M阶段。这些数据表明，代谢差异是容易检测由该方法甚至密切相关的亚群之间。 <img alt="图1" src="/files/ftp_upload/55011/55011fig1.jpg" /> 图1: 实验设计。工作流程准备的菜，模拟分类和排序的额外的亚群CTS。盘和模拟分类提取物的菜和模拟分类提取物的验证实验使用的MID进行比较试验，由于排序可能发生的变化。细胞的复杂群是根据感兴趣的细胞类型的标记排序，然后萃取。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55011/55011fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55011/55011fig2.jpg" /> 图2:对直接提取的MID的验证。 （ 一 ）13 C散点图和菜中15 N富集和模拟分类样本。每个点代表一个复制。一式三份都加入了与线。 （ 二 ）谷氨酸13 C- 15 N的MID。 （C）13 C乳酸移动互联网设备。误差棒为一式三份measu的标准偏差rements。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55011/55011fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55011/55011fig3.jpg" /> 图3: 在HeLa细胞中的细胞周期阶段之间代谢的差异。胞苷在G 1 -G 0和SG 2 -M细胞不同标记。 （A）胞苷13 C富集在G中1 -G 0和SG 2 -M期细胞周期。虚线表示从天然同位素质谱碳富集。如图G中1 -G 0阵列的情节和SG 2 -M阶段（B）胞苷的MID。误差线是三次测量的标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55011/55011fig3large.jpg" target="_blank">请点击此处到v</a>IEW这个数字的放大版本。

Watch this Scientific Journal Video about A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing at JoVE.com

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

a-method-for-measuring-metabolism-sorted-subpopulations-complex-cell

Nanyang Technological University

Research

JoVE Journal

Biochemistry

8.2K 次观看.  Karolinska Institutet. This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

视频: A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer tissue with minor or no modification, allowing the dietary routing between different trophic levels. However, the FA trophic marker approach is still hampered by the limited knowledge in lipid metabolism of the soil fauna. This study used entirely labelled palmitic acid (13C16:0, 99 atom%) as a tracer in fatty acid metabolism pathways of two widespread soil Collembola, Protaphorura fimata and Heteromurus nitidus. In order to investigate the fate and metabolic modifications of this precursor, a method of isotopologue profiling is presented, performed by mass spectrometry using single ion monitoring. Moreover, the upstream laboratory feeding experiment is described, as well as the extraction and methylation of dominant lipid fractions (neutral lipids, phospholipids) and the related formula and calculations. Isotopologue profiling does not only yield the overall 13C enrichment in fatty acids derived from the 13C labeled precursor but also produces the pattern of isotopologues exceeding the mass of the parent ion (i.e., the FA molecular ion M+) of each labeled FA by one or more mass units (M+1, M+2, M+3, etc.). This knowledge allows conclusions on the ratio of dietary routing of an entirely consumed FA in comparison to de novo biosynthesis. The isotopologue profiling is suggested as a useful tool for evaluation of fatty acid metabolism in soil animals to disentangle trophic interactions.

Fatty Acid 13C Isotopologue Profiling Provides Insight into Trophic Carbon Transfer and Lipid Metabolism of Invertebrate Consumers

The fatty acid trophic marker approach, i.e., the assimilation of fatty acids as entire molecule and transfer into consumer tissue with no or minor modification, is hampered by knowledge gaps in fatty acid metabolism of small soil invertebrates. Isotopologue profiling is provided as a valuable tool to disentangle trophic interactions.

脂肪酸13C Isotopologue 分析提供了对无脊椎动物消费者营养碳转移和脂质代谢的洞察力

Fatty acids (FAs) are useful biomarkers in food web ecology because they are typically assimilated as a complete molecule and transferred into consumer ...

科研

生物化学

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

通过反相液相-质谱法对无细胞蛋白合成代谢的绝对定量

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

使用代谢标记和质谱法测量衰老和非分裂培养细胞中的蛋白质周转率

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Workflow Based on the Combination of Isotopic Tracer Experiments to Investigate Microbial Metabolism of Multiple Nutrient Sources

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods substantially contributes to providing extensive knowledge of the metabolism of microorganisms growing in chemically defined media containing unique nitrogen and carbon sources. In contrast, the management through metabolism of multiple nutrient sources, despite their broad presence in natural or industrial environments, remains virtually unexplored. This situation is mainly due to the lack of suitable methodologies, which hinders investigations.
We report an experimental strategy to quantitatively and comprehensively explore how metabolism operates when a nutrient is provided as a mixture of different molecules, i.e., a complex resource. Here, we describe its application for assessing the partitioning of multiple nitrogen sources through the yeast metabolic network. The workflow combines information obtained during stable isotope tracer experiments using selected 13C- or 15N-labeled substrates. It first consists of parallel and reproducible fermentations in the same medium, which includes a mixture of N-containing molecules; however,a selected nitrogen source is labeled each time. A combination of analytical procedures (HPLC, GC-MS) is implemented to assess the labeling patterns of targeted compounds and to quantify the consumption and recovery of substrates in other metabolites. An integrated analysis of the complete dataset provides an overview of the fate of consumed substrates within cells. This approach requires an accurate protocol for the collection of samples&#8211;facilitated by a robot-assisted system for online monitoring of fermentations&#8211;and the achievement of numerous time-consuming analyses. Despite these constraints, it allowed understanding, for the first time, the partitioning of multiple nitrogen sources throughout the yeast metabolic network. We elucidated the redistribution of nitrogen from more abundant sources toward other N-compounds and determined the metabolic origins of volatile molecules and proteinogenic amino acids.

This protocol describes an experimental procedure to quantitatively and comprehensively investigate the metabolism of multiple nutrient sources. This workflow, based on a combination of isotopic tracer experiments and an analytical procedure, allows the fate of consumed nutrients and the metabolic origin of molecules synthetized by microorganisms to be determined.

基于同位素示踪实验结合的工作流研究多营养源的微生物代谢

Studies in the field of microbiology rely on the implementation of a wide range of methodologies. In particular, the development of appropriate methods ...

生物工程

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

在开发和成年阶段的中介代谢通量的稳定同位素分析线虫</em

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

生物学

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.

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.

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 Analysis of Drosophila melanogaster Larval and Adult Brains

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs provides a tissue-level understanding of energy utilization that cannot be captured when analyzing primary cells and cell lines. While this analysis is ex vivo, it allows for the measurement from a number of specialized cells working together to perform a function in one tissue and more closely models the in vivo organ. Metabolic reprogramming has been observed in many neurological diseases, including neoplasia, and neurodegenerative diseases. This protocol was developed to assist the D. melanogaster community&#39;s investigation of metabolism in neurological disease models using a commercially available metabolic analyzer. Measuring metabolism of whole brains in the metabolic analyzer is challenging due to the geometry of the brain. This analyzer requires samples to remain at the bottom of a 96-well plate. Cell samples and tissue punches can adhere to the surface of the cell plate or utilize spheroid plates, respectively. However, the spherical, three-dimensional shape of D. melanogaster brains prevents the tissue from adhering to the plate. This protocol requires a specially designed and manufactured micro-tissue restraint that circumvents this problem by preventing any movement of the brain while still allowing metabolic measurements from the analyzer&#39;s two solid-state sensor probes. Oxygen consumption and extracellular acidification rates are reproducible and sensitive to a treatment with metabolic inhibitors. With a minor optimization, this protocol can be adapted for use with any whole tissue and/or model system, provided that the sample size does not exceed the chamber generated by the restraint. While basal metabolic measurements and an analysis after a treatment with mitochondrial inhibitors are described within this protocol, countless experimental conditions, such as energy source preference and rearing environment, could be interrogated.

Metabolic Analysis of Drosophila melanogaster Larval and Adult Brains

We present a protocol for measuring oxygen consumption and extracellular acidification in Drosophila melanogaster larval and adult brains. A metabolic analyzer is utilized with an adapted and optimized protocol. Micro-tissue restraints are a critical component of this protocol and were designed and created specifically for their use in this analysis.

果蝇幼虫和成年脑的代谢分析

This protocol describes a method for measuring the metabolism in Drosophila melanogaster larval and adult brains. Quantifying metabolism in whole organs ...

一种用于在复杂的细胞群落稳定性同位素示踪排序亚群测量方法代谢

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在开发和成年阶段的中介代谢通量的稳定同位素分析线虫

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

果蝇幼虫和成年脑的代谢分析