Name: simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures
Uploaded: 2020-05-09T13:30:00.000+00:00
Duration: 10 min 38 s
Description: Watch this Scientific Journal Video about Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures at JoVE.

这项工作得到欧洲联盟根据《2007-2013年波兰东部业务方案发展》获得欧洲联盟区域发展基金的支持（POPW.01.03.00-06-003/09-00），由卢布林约翰·保罗二世天主教大学生物技术和环境科学学院（波兰国家科学中心为伊洛娜·萨多克提供青年科学家赠款）， OPUS13 （2017/25/B/NZ4/01198 马格达莱娜·斯塔尼谢夫斯卡，原则调查员）。 作者感谢JPY CUL跨学科研究中心氧化应激实验室的阿格涅什卡·奇比尔教授分享细胞培养和蛋白质定量设备。

1. 准备标准 3NT、Kyn、3HKyn、3HAA、XA 标准库存解决方案
<ol><li>以最高精度（每瓶0.3毫克）在小瓶中称量试剂。为了提高准确性，请放大试剂，根据步骤 1.2 调整溶剂的体积。</li>
	<li>溶解溶剂在溶剂的300μL，以获得1克/升的库存溶液。溶解3NT在1%（v/v）的微酸（FA）在水中;金，3HAA和XA在二甲基硫化物（DMSO）;3HKyn 在水中酸化到 pH 2.5 与盐酸 （HCL）. 警告:3HAA刺激眼睛、鼻子、喉咙和肺部。吸入、接触皮肤和吞咽时有害。佩戴适当的防护服，即手套和面罩。</li>
	<li>紧闭小瓶，放在超声波浴缸中1分钟，以加速溶解。 注意:将库存解决方案存储在 -20 °C，并最大限度地减少由于库存解决方案不稳定造成的冻结/解冻（最大 3 个周期）。</li>
</ol>2. 木炭处理文化媒介的制备
<ol><li>在一根管子中，重280毫克的活性炭，并加入5 mL的液体介质，准备培养感兴趣的细胞。</li>
	<li>在室温下（将速度设置为 50 振荡/分钟），用锯锯摇杆上的介质和木炭摇动管 2 小时。接下来，将管子在 6000 x g 下离心 15 分钟。</li>
	<li>从离心机中取出管子，小心收集超纳坦，而不会干扰沉积物。如有必要，重复离心，以去除所有木炭残留物。</li>
	<li>使用 0.45 μm 注射器过滤器过滤超强剂。</li>
	<li>通过在LC-MS上运行第 6.3.2 步中描述的试点样本，确保木炭预处理培养基被剥夺了 kynurenine 痕迹。否则，重复步骤 2.1-2.4。 注:如果完整的培养基最初不含基努他宁，则可能会省略使用木炭的净化步骤。在这种情况下，使用通常用于细胞培养的完整介质准备校准标准和质量控制样品。</li>
	<li>将准备好的介质存储在4°C，直到分析。</li>
</ol>3. 制作校准解决方案和校准曲线
<ol><li>用 0.75 μL 的 0.1 g/L 3NT 溶液喷涂木炭预处理培养基，并添加四个标准（3HKyn、3HAA、Kyn、XA）至少六种不同浓度，以覆盖校准范围。将每个样品的最终体积保持在 150μL. 漩涡良好。使用 1.5 mL 离心管进行样品准备。 注:3HKyn建议校准点:0.018、0.045、0.22、1.12、2.23、4.46 μmol/L:Kyn: 0.0096， 0.048， 0.24， 1.20， 2.40， 3.84 μmol/L;3HAA: 0.033， 0.16， 0.65， 3.27， 6.53， 13.06 μmol/L;XA: 0.019， 0.13， 0.49， 1.22， 3.65， 4.87 μmol/L.</li>
	<li>在每个管子中加入 150 μL 的冷甲醇（保持在 -20 °C），其中含有 1% （v/v） 的甲醇，用于样品脱蛋白。紧紧地关闭管子和漩涡。</li>
	<li>在-20°C下孵化样品40分钟。</li>
	<li>在 4 °C 下将样品在 14，000 x g 下离心 15 分钟。 从离心机中取出管子。将超纳丁收集到新管中。请勿干扰沉积物。</li>
	<li>使用自动移液器将 270μL 的超级麻醉剂转移到玻璃瓶中。将小瓶放入蒸发器中，轻轻蒸发，直到干燥。使用适当的程序对水/甲醇分数蒸发挥发性成分。不要应用温度高于40°C，避免过度干燥。 注:平底玻璃瓶，即色谱瓶，与塑料圆锥管相比，可促进更快的蒸发和更高的回收。</li>
	<li>30分钟后，检查蒸发状态。如有必要，继续蒸发（例如，额外增加10分钟）。避免过度干燥。</li>
	<li>从蒸发器中取出小瓶。将每个样品在 60 μL 中重新构合 0.1% （v/v） 水中的微酸。将溶剂加入含有残留物质的瓶中。紧紧关闭小瓶和漩涡井。</li>
	<li>将样品转移到 1.5 mL 管中，在 14，000 x g 下旋转 15 分钟，在 4 °C 下分离沉淀的蛋白质。</li>
	<li>在不干扰蛋白质颗粒的情况下，使用带自动移液器的锥形玻璃插入将超级纳坦转移到色谱小瓶中。紧紧关闭小瓶。</li>
	<li>检查插入瓶中是否存在气泡，如有必要，将其取出（例如，通过涡旋）。</li>
	<li>将含有样品的色谱小瓶转移到LC自动取样器中。记录放置在自动取样器托盘中的样品的位置。</li>
</ol>4. 编制质量控制 （QC） 样品
<ol><li>用0.75微升的0.1克/升3NT溶液和四种基努他宁（3HKyn、3HAA、Kyn、XA）的标准，在从校准曲线的线性范围内选择的浓度中，将木炭预处理培养基（在1.5毫升离心管中制备）尖峰。将样品的最终体积保持在 150 μL。 注:如果适用，请准备几个不同浓度的QC样品，这些样品属于校准曲线的线性范围。</li>
	<li>遵循第 3 节中描述的协议（步骤 3.2-3.11）。</li>
</ol>5. 建立LC-MS系统
<ol><li>准备移动相溶剂:溶剂A:超纯水中的20毫升/升铵（pH 4.3经氨酸调节）:溶剂 B: 100% 丙酮三星。 注意:仅使用波罗西酸玻璃瓶。在重新灌装之前用超纯水冲洗瓶子。不要使用用洗涤剂清洗的瓶子。 警告:丙酮三星会导致严重的健康影响或死亡。它很容易被热点燃。丙酮液体和蒸汽会刺激眼睛、鼻子、喉咙和肺部。<ol>
<li>将晶体试剂（15.77克）溶解在玻璃瓶中的50毫升超纯净水中，准备5摩尔/L铵汤溶液。搅拌，直到所有残留物溶解。过滤 0.45 μm 膜以清除任何残留碎片（例如，使用尼龙注射器过滤器）。将库存解决方案存储在4°C。</li>
		<li>准备溶剂A通过添加4毫升5摩尔/升铵在水中到980毫升的超纯水在琥珀色玻璃瓶和搅拌井。浸入搅拌棒，将带溶剂的瓶子放在磁性搅拌器上。将 pH 电极浸入溶液中，并在搅拌下控制 pH。添加氨酸库存溶液 （98%-100%）使用带 0.2 mL 尖端的自动移液器获得 pH 4.3，用超纯水将音量调整至 1 L，搅拌良好。 注意:每周至少准备一次溶剂以防止任何微生物生长。 警告:福密酸 （FA） 吸入时有毒，导致皮肤灼伤和眼睛损伤。在烟雾罩下工作;戴防护手套和外套。</li>
		<li>通过 0.22 μm 膜（例如尼龙注射器过滤器）过滤所有溶液，以清除任何残留碎片。可选地，使用专用于LC溶剂储层的溶剂进气口过滤器，以保护系统免受传入颗粒的射入。</li>
	</ol></li>
	<li>启动LC-MS控制和数据采集软件。</li>
	<li>使用移动相清除LC系统以去除气泡，并使所有溶剂通道都处于最佳状态。</li>
	<li>组合一个守卫柱，以保护分析列免受堵塞。</li>
	<li>用 100% 丙酮三星冲洗护栏和分析柱（C18、2.1 x 100 mm、1.8 μm）约 30 分钟，然后用 100% 溶剂 A 冲洗，直到观察到柱子中的稳定压力。使用控制软件控制系统性能。</li>
	<li>在数据采集软件中设置相应的LC参数。<ol>
<li>设置梯度方案:0-8分钟溶剂B 0%:8-17 分钟溶剂 B 0-2%:17-20分钟溶剂B 2%:20-32 分钟溶剂 B 10-30%:32-45 分钟溶剂 B 0%。</li>
		<li>将移动相流速设置为 0.15 mL/min，总分析运行时间为 45 分钟，柱温为 +40 °C，喷射体积为 10μL。</li>
	</ol></li>
	<li>设置质谱探测器的采集参数。<ol>
<li>应用电化参数:单离子监测（SIM）、正电化、雾化压力50 psi、+350°C干燥气体温度、12升/分钟的干燥气体流量和5500 V毛细管电压。</li>
		<li>选择分析监测离子:3HKyn m/z 225.0（扫描周期:2-6分钟，碎块电压:100 V）:用于 Kyn m/z 209.0（扫描周期:6-12 分钟，碎块电压:80 V）:3HAA m/z 154.0（扫描周期:12-18 分钟，碎块电压:80 V）:3NT m/z 227.0（扫描周期:18-23分钟，碎块电压:100 V）:XA m/z 206.0（扫描周期:23-45分钟，碎块电压:100 V）。</li>
	</ol></li>
	<li>构建工作列表并在LC系统上运行示例。</li>
	<li>首先，在分析实验样品之前，至少在三个样本中运行一个空白样本（溶剂A），然后是一个QC样本。</li>
	<li>打开数据分析软件并加载为 QC 样本获得的结果。</li>
	<li>检查色谱上分析峰的位置。当信号移到预期位置之外时，请调整时间门以进行适当的分析信号收集。有关信号集成的说明，请参阅软件手册。</li>
</ol>6. 构建校准曲线
<ol><li>在数据采集软件中，将校准标准添加到工作列表中，并在三次运行标准。</li>
	<li>分别在约4.4分钟、10分钟、16分钟、21分钟和30分钟的保留时间内，对应3HKyn、Kyn、3HA、3NT和XA的峰值区域进行集成和测量。</li>
	<li>使用电子表格程序为每个代谢物构建单个校准曲线。<ol>
<li>要创建校准图，则绘制 3NT 峰值区域中分析峰值区域与分析物浓度的比率值。</li>
		<li>分析空白样本（仅用3NT喷射的炭预处理培养基），确保介质中没有研究分析物的痕迹。否则，从每个校准点减去所获得的价值。</li>
		<li>检查每个校准曲线的线性。<ol>
<li>单独地，对于每个分析器，创建一个斜面拦截线性方程（y = ax +b），其中 y 对应分析峰值区域与 3NT 峰值区域的比例，a是斜率，x是分析浓度（μmol/L），b 表示曲线拦截。绘制图形"y"与"x"将生成校准曲线。</li>
			<li>在图表中添加线性趋势线，在图表上显示校准曲线方程和回归系数。确保回归系数为+0.990。如果校准标准不匹配，再次准备和/或分析这些标准。可选地更改校准曲线的浓度范围。</li>
		</ol>
</li>
		<li>在分析实验样品之前，先分析 QC 样本以检查系统性能。如果不兼容（±参考值偏离 20%），则准备新的校准曲线。</li>
	</ol>
</li>
</ol>7. 体外细胞培养和样本收集，供分析
<ol><li>板研究的癌细胞（MDA-MB-231或SK-OV-3）在DMEM（杜尔贝科的改良鹰的介质）含有4.5克/升的ᴅ葡萄糖， 10% （v/v） 胎儿牛血清 （FBS）， 2 mmol/L ʟ-谷氨酰胺和 1 U 青霉素/链霉素和培养在 37 °C 在 5% CO2 的加湿大气中， 以扩大他们为实验.以 80% 的汇合率通过细胞。</li>
	<li>使用trypsin将细胞从培养皿中分离出，在12口井板上进行实验的种子，密度为每口井0.3×106 个细胞，并放置在孵化器中过夜。</li>
	<li>第二天，吸气培养基，并添加新鲜的DMEM，无论是否添加研究的化合物，这取决于实验设计。</li>
	<li>48小时后，从细胞上方（约500μL）收集介质到1.5 mL管中。离心机在14，000 x g 5分钟，以消除任何细胞碎片。收集超纳坦并储存在-80°C，直到分析。</li>
	<li>将细胞颗粒从步骤 7.4 中保存以进行蛋白质估计。将样品冻结在-20°C。 注:从不同油井获得的介质结果应标准化为总蛋白质含量，以考虑单个油井中细胞数的变异性。蛋白质浓度可以按照第 9 步进行评估。</li>
</ol>8. 为LC-MS分析准备样品
<ol><li>在室温下解冻冷冻样品，混合均好。将培养基的149.25μL转移到离心管（1.5 mL）中。</li>
	<li>添加 3NT 解决方案 0.1 克/升的 0.75 微升（内部标准）。关闭管子和漩涡很好。</li>
	<li>添加 150 μL 的冰镇在 -20 °C 甲醇含有 1% （v/v） FA 样品去蛋白化。关闭管子和漩涡很好。</li>
	<li>继续第 3 节（步骤 3.3-3.11）中描述的样品准备。 注:一些癌细胞，如SK-OV-3，将大量的Kyn分泌到培养基中。要将数据放入校准曲线的线性范围内，需要对样品进行大约 100 倍的稀释。在这种情况下，在水中用 0.1% （v/v） FA 稀释部分样品，并分析未稀释的样品。校准曲线标准的参考样本应在100倍稀释的完整培养介质中编写。或者，在校准曲线中添加更多点（如果达到适当的溶解度和线性），使其调整到实验样本中 Kyn 的浓度水平。</li>
	<li>分析第 5 节中描述的 LC-MS 样本。构建一个工作列表，并将每个样本运行三次。</li>
	<li>工作表完成后，测量分析的峰值区域。</li>
	<li>使用可用的软件生成电子表格和获取的数字数据。</li>
	<li>在一个电子表格列中计算 3NT 峰值区域上的分析峰值区域比率。</li>
	<li>使用专用于每个分析量（从步骤 6.3.）的单个线性校准方程来计算实验样本中存在的分析物浓度。</li>
</ol>9. 评估蛋白质含量和数据正常化
<ol><li>将细胞颗粒从步骤 7.5 中重新悬生，在 1.5 mL 管中使用 100μL 磷酸盐缓冲盐水 （PBS）。 注:在950mL超纯水中溶解8克氯化钠、0.2克氯化钾、1.44克磷酸二酸钠、磷酸二氢钾，准备PBS溶液。搅拌好。用盐酸（滴式）将pH调整为7.4。用超纯水调整体积高达 1 L。搅拌好，直到均匀。 注意:盐酸具有高度腐蚀性，必须采取适当的安全防范措施。与人的皮肤接触可导致发红、疼痛、皮肤灼伤。在烟雾罩下工作;戴防护手套和外套。</li>
	<li>将样品冷冻在-20°C，然后在冰上解冻。重复此步骤3倍。</li>
	<li>在 4 °C 下将样品在 14，000 x g 下离心 15 分钟。 将超纳托收集到新管中。不要打扰颗粒。</li>
	<li>用PBS稀释超常剂10倍。</li>
	<li>构造一个校准曲线，每个井范围的蛋白质为 0 到 2 μg。<ol>
<li>准备牛血清白蛋白 （BSA） 标准溶液进行校准。溶解 1.23 μg 的 BSA 在 1 mL 的超纯水和涡井。</li>
		<li>在 96 井板上，装载不同数量的 BSA 标准解决方案（1.23 μg/mL）:0 μL、2 μL、4 μL、5 μL、7 μL、10 μL、15 μL、20 μL。</li>
		<li>用 PBS 填充油井，最终体积为 50 μL。</li>
	</ol>
</li>
	<li>加载 10 - 15 μL 细胞从步骤 9.4 在同一 96 井板上解压。用 PBS 填充油井，达到 50 μL 的最终体积。 注:如有必要，请调整每口井中细胞解液的量，以适应校准曲线的线性范围。将每个油井的样本最终体积保持在 50μL。</li>
	<li>在每个井中加入 200μL 的布拉德福德试剂（用超纯水稀释 5 次）。</li>
	<li>在室温下将96口井的馅饼孵育至少5分钟。将板插入微板读取器。测量λ=595nm的吸收量。</li>
	<li>使用电子表格程序构建校准曲线。绘制 BSA 量 （μg） 与吸收量。添加线性趋势线，在图表上显示校准曲线方程和回归系数。</li>
	<li>使用线性方程（y = ax +b，其中y对应于λ = 595 nm 的吸收，a是斜率，x是蛋白质含量（μg），b 指曲线截获）来计算样品中的蛋白质量。 注:在计算中考虑样品稀释因子。</li>
	<li>使用估计的蛋白质含量来使每1毫克蛋白质样本中的基努他宁量正常化。为此，将第 8.9 步确定的 kynurenine 浓度与第 9.10 步的总蛋白质含量分开。</li>
</ol>

<ol>
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作者没有什么可透露的。

本文提出了一个详细的LC-SQ协议，用于同时量化四个主要的Trp代谢物（3HKyn，Kyn，3HAA，XA），这些代谢物是用 体外 培养的人类癌细胞的介质测量的。特别注意样品的准备、色谱/质谱过程和数据解释，这是分析中最重要的一点。
一般来说，LC-MS 分析由于灵敏度高，需要对二手材料的严格性和纯度进行最高标准的协议。它是指在整个标准程序中应用整齐清洁和正确洗涤的?...

基努琳通路 （KP） 是人类细胞中色氨酸 （Trp） 催化的主要途径。在肝外细胞中，印度胺-2，3-二氧酶（IDO-1）是第一种限制KP酶，并将Trp转化为N-形成基核素1。KP 内的进一步步骤会生成其他次要代谢物，即具有各种生物特性的基努他宁。Kynurenine （Kyn） 是第一个稳定的 Trp 催化物， 显示有毒性质和调节细胞事件后， 结合到阿丽尔碳氢化合物受体 （AhR）2.随后，Kyn被自发地或在酶介质过程中转化为几个分子，产生诸如3-羟基核素（3HKyn）、炭酸（AA）、3-羟基苯甲酸（3-HAA）、基努雷尼酸（Kyna）和Xanthurenic酸（XA）等代谢物。另一种下游代谢物，2-氨基-3-卡博基霉素酸-6-半醛（ACMS），经历非酶循环奎诺利尼酸（QA）或野餐酸（PA）1.最后，QA被进一步转化为烟酰胺-腺苷二核苷酸（NAD+）3，KP端点代谢物，是一个重要的酶共同因子。一些基努肾上腺素具有神经保护特性，如Kyna和PA，而其他，即3HAA和3HKyn，是有毒的4。由3HKyn形成的Xanthurenic酸具有抗氧化和血管电离特性5。XA在老化的镜片中积累，导致上皮细胞6的凋亡。KP在20世纪中叶被描述，当它参与各种疾病被证明时，它得到了更多的关注。这种代谢途径的增加和一些kynurenine的积累调节免疫反应，并与不同的病理条件，如抑郁症，精神分裂症，脑病，艾滋病毒，痴呆症，肌萎缩性侧索硬化症，疟疾，阿尔茨海默氏症，亨廷顿舞蹈症和癌症4，7。在肿瘤微环境和癌细胞2，8中观察到Trp新陈代谢的一些变化。此外，基努雷宁被认为是有希望的疾病标记9。在癌症研究中，体外细胞培养模型已建立并广泛应用于抗癌药物10的药前评价。Trp代谢物由细胞分泌到培养基中，可以测量以评估基努艾琳通路的状态。因此，需要开发适当的方法，以简单、灵活和可靠的协议，在各种生物标本中同时检测尽可能多的KP代谢物。
在本文中，我们描述了一个协议，用于同时确定四个基努艾琳通路代谢物:Kyn、3HKyn、3HAA和XA，由LC-SQ在从癌细胞中收集的培养基中。在现代分析方法中，液相色谱13、14、15、16优先于同时检测和量化单个色氨酸催化物，而使用Erlich试剂11、12进行生化非特异性检测。目前，人类标本中有许多用于基努他宁测定的方法，主要基于带有紫外线或荧光检测仪13、17、18、19的液相色谱。液相色谱与质谱探测器 （LC-MS） 似乎更适合此类分析，因为其灵敏度较高（检测范围较低）、选择性和可重复性。
Trp代谢物已经确定在人类血清，血浆和尿液13，20，21，22，23，但是，其他生物标本的方法，如细胞培养介质也是需要的。此前，LC-MS用于培养人类胶质瘤细胞、单核细胞、支气管细胞或用干扰素伽马（IFN-γ）治疗的星形细胞（IFN-γ）24、25、26后收集的介质中的Trp衍生化合物。目前，需要新的验证协议，可以评估不同培养基、细胞和癌症模型中使用的治疗方法中的几个代谢物。
开发方法的目的是量化（在一次分析运行内）四个主要的基努雷宁，可以指示KP的异常。这里介绍的是我们最近出版的协议的关键步骤，定量LC-SQ分析选定的有意义的基努雷宁使用一个内部标准（3-硝基罗辛，3NT）在从 体外 培养的人类癌细胞27收集的介质。据我们所知，这是第一个LC-SQ协议，用于在从 体外 生长细胞获得的培养基中同时量化3HKyn、3HAA、Kyn和XA。经过一些修改后，该方法可以进一步应用于研究Trp代谢在更广泛的细胞培养模型中的变化。

<table><tbody><tr><td>3-hydroksy-DL-kynurenina</td><td>Sigma-Aldrich</td><td>H1771</td><td></td></tr><tr><td>3-hydroxyanthranilic acid</td><td>Sigma-Aldrich</td><td>148776</td><td>97%</td></tr><tr><td>3-nitro-L-tyrosine</td><td>Sigma-Aldrich</td><td>N7389</td><td></td></tr><tr><td>Acetonitrile</td><td>Supelco</td><td>1.00029</td><td>hypergrade for LC-MS LiChrosolv</td></tr><tr><td>Activated charcoal</td><td>Supelco</td><td>05105</td><td>powder</td></tr><tr><td>Analytical balance</td><td>Ohaus</td><td></td><td></td></tr><tr><td>Analytical column</td><td>Agilent Technologies</td><td>959764-902</td><td>Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 &amp;micro;m)</td></tr><tr><td>Ammonium formate</td><td>Supelco</td><td>7022</td><td>eluent additive for LC-MS, LiChropur, &amp;ge;99.0%</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>1001887398</td><td></td></tr><tr><td>Caps for chromatographic vials</td><td>Agilent Technologies</td><td>5185-5820</td><td>blue screw caps,PTFE/red sil sepa</td></tr><tr><td>Cell culture dish</td><td>Nest</td><td>704004</td><td>polystyrene, non-pyrogenic, sterile</td></tr><tr><td>Cell culture plate</td><td>Biologix</td><td>07-6012</td><td>12-well, non-pyrogenic, non-cytoxic, sterille</td></tr><tr><td>Cell incubator</td><td>Thermo Fisher Scientific</td><td></td><td>HERAcell 150i Cu</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td>model 5415R</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td></td><td>model 5428</td></tr><tr><td>Centrifuge tubes</td><td>Bionovo</td><td>B-2278</td><td>Eppendorf type, 1.5 mL</td></tr><tr><td>Centrifuge tubes</td><td>Bionovo</td><td>B-3693</td><td>Falcon type, 50 mL, PP</td></tr><tr><td>Chromatographic data acqusition and analysis software</td><td>Agilent Technologies</td><td></td><td>LC/MSD ChemStation (B.04.03-S92)</td></tr><tr><td>Chromatographic insert vials</td><td>Agilent Technologies</td><td>9301-1387</td><td>100 &amp;micro;L</td></tr><tr><td>Chromatographic vials</td><td>Agilent Technologies</td><td>5182-0714</td><td>2 mL, clear glass</td></tr><tr><td>Dimethyl sulfoxide</td><td>Supelco</td><td>1.02950</td><td>Uvasol</td></tr><tr><td>Dubelcco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td>PAN Biotech</td><td>P04-41450</td><td></td></tr><tr><td>Dual meter pH/conductivity</td><td>Mettler Toledo</td><td></td><td>SevenMulti</td></tr><tr><td>Evaporator</td><td>Genevac</td><td></td><td>model EZ-2.3 Elite</td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich</td><td>F9665</td><td></td></tr><tr><td>Formic acid</td><td>Supelco</td><td>5.33002</td><td>for LC-MS LiChropur</td></tr><tr><td>Glass bottle for reagents storage</td><td>Bionovo</td><td>S-2070</td><td>50 mL, clear glass</td></tr><tr><td>Guard column</td><td>Agilent Technologies</td><td>959757-902</td><td>Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 &amp;micro;m)</td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>1.00317.1000</td><td></td></tr><tr><td>L-kynurenine</td><td>Sigma-Aldrich</td><td>K8625</td><td>&amp;ge;98% (HPLC)</td></tr><tr><td>Magnetic stirrer</td><td>Wigo</td><td></td><td>ES 21 H</td></tr><tr><td>Microbalance</td><td>Mettler Toledo</td><td></td><td>model XP6</td></tr><tr><td>Milli-Q system</td><td>Millipore</td><td>ZRQSVPO30</td><td>Direct-Q 3 UV with Pump</td></tr><tr><td>Quadrupole mass spectrometer</td><td>Agilent Technologies</td><td>G1948B</td><td>model 6120</td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Adrich</td><td>048M4874V</td><td></td></tr><tr><td>Plate reader</td><td>Bio Tek</td><td></td><td>Synergy 2 operated by Gen5</td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>1.04936.1000</td><td></td></tr><tr><td>Potassium dihydrogen phosphate</td><td>Merck</td><td>1.05108.0500</td><td></td></tr><tr><td>Protein Assay Dye Reagent Concentrate</td><td>BioRad</td><td>500-0006</td><td></td></tr><tr><td>See-saw rocker</td><td>Stuart</td><td>SSL4</td><td></td></tr><tr><td>Serological pipette</td><td>Nest</td><td>326001</td><td>5 mL, polystyrene, non-pyrogenic, sterile</td></tr><tr><td>Sodium chloride</td><td>Sigma-Aldrich</td><td>7647-14-5</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Merck</td><td>1.06346.1000</td><td></td></tr><tr><td>Solvent inlet filter</td><td>Agilent Technologies</td><td>5041-2168</td><td>glass filter, 20 &amp;mu;m pore size</td></tr><tr><td>Solvent reservoir (for LC-MS)</td><td>Agilent Technologies</td><td>9301-6524</td><td>1 L, clear glass</td></tr><tr><td>Solvent reservoir (for LC-MS)</td><td>Agilent Technologies</td><td>9301-6526</td><td>1 L, amber glass</td></tr><tr><td>Spreadsheet program</td><td>Microsoft Office</td><td></td><td>Microsoft Office Excel</td></tr><tr><td>Stir bar</td><td>Bionovo</td><td>6-2003</td><td>teflon coated</td></tr><tr><td>Syringe filters for culture medium filtration</td><td>Bionovo</td><td>7-8803</td><td>regenerated cellulose, &amp;Oslash; 30 mm, 0,45 &amp;micro;m</td></tr><tr><td>Syringe filters for mobile phase components filtration</td><td>Bionovo</td><td>6-0018</td><td>nylon, &amp;Oslash; 30 mm, 0,22 &amp;micro;m</td></tr><tr><td>Tissue culture plates</td><td>VWR</td><td>10062-900</td><td>96-wells, sterille</td></tr><tr><td>Trypsin-EDTA Solution</td><td>Sigma-Aldrih</td><td>T4049-100 mL</td><td></td></tr><tr><td>Ultra-High Performance Liquid Chromatography system</td><td>Agilent Technologies</td><td>G1367D, G1379B, G1312B, G1316C</td><td>1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C)</td></tr><tr><td>Ultrasound bath</td><td>Polsonic</td><td>104533</td><td>model 6D</td></tr><tr><td>Vortex</td><td>Biosan</td><td></td><td>model V-1 plus</td></tr><tr><td>Xanthurenic acid</td><td>Sigma-Aldrich</td><td>D120804</td><td>96%</td></tr></tbody></table>

simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures

基努雷宁通路和色氨酸卡塔博利特称为基努雷宁已经受到越来越多的关注，因为他们参与免疫调节和癌症生物学。 体外 细胞培养分析通常用于了解不同色氨酸卡托邦细胞在疾病机制中的贡献以及用于测试治疗策略。富含分泌代谢物和信号分子的细胞培养介质反映了色氨酸代谢和其他细胞事件的状态。需要在复杂的细胞培养介质中可靠量化多种基努他宁的新协议，以便对多个样本进行可靠和快速的分析。这可以通过液体色谱与质谱学相结合来完成。这种强大的技术用于许多临床和研究实验室的代谢物的定量，可用于测量基努他宁。
这里介绍的是使用液相色谱与单四足质谱（LC-SQ）同时确定四个基努他宁，即基努雷宁，3-羟基核素，3-羟基苯甲酸，和从 体外 培养癌细胞中收集的介质中的黄曲霉素酸。SQ探测器使用简单，与其他质谱仪相比更便宜。在 SQ-MS 分析中，根据样品的特定质量与电荷比（m/z）生成并分离多个离子，然后使用单离子监控 （SIM） 模式进行检测。
本文注重报告方法的优点，指出了一些薄弱环节。它侧重于LC-SQ分析的关键要素，包括样品准备以及色谱和质谱分析。还讨论了质量控制、方法校准条件和矩阵效应等问题。我们描述了一个简单的应用3硝基酪氨酸作为一个模拟标准的所有目标分析。经人类卵巢癌和乳腺癌细胞实验证实，建议的LC-SQ方法产生可靠的结果，并可进一步应用于其他 体外 细胞模型。

LC-MS在生物活性分子的量化方面具有无可争辩的优势，尽管复杂标本的某些成分会导致所谓的基质效应，并损害分析物的电位化。它导致离子抑制或离子增强，严重降低精度，并影响LC-MS的检测/量化极限，这被认为是该方法的"弱"点。在我们的协议中，离子是由正模式下的电喷雾电离子化（ESI）产生的，这也用于Trp代谢物测定13的其他研究。然而，与大气压力化学电化<sup class="xref...

Watch this Scientific Journal Video about Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures at JoVE.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

这里描述的是一个协议，用于确定从液体色谱分析的癌细胞培养物中收集的介质中产生的四种不同的色氨酸代谢物（金黄素、3-羟基核素、Xanthurenic酸、3-羟基苯甲酸）。

从癌细胞培养物中收集的介质光谱分析的选定金核素的同步量化

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

simultaneous-quantification-selected-kynurenines-analyzed-liquid

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

3.3K 次观看.  The John Paul II Catholic University of Lublin. 这里描述的是一个协议，用于确定从液体色谱分析的癌细胞培养物中收集的介质中产生的四种不同的色氨酸代谢物（金黄素、3-羟基核素、Xanthurenic酸、3-羟基苯甲酸）。

视频: Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

质谱法和基于发光的-途径表征阶段我代谢能力的<em&gt;体外</em&gt;细胞培养

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

科研

生物化学

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

生物工程

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

HPLC-基于化验，以监测外核苷酸/核苷代谢的人类慢性淋巴细胞白血病细胞

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

化学

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

解吸电喷雾电离质谱成像的生物组织

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

A High-performance Liquid Chromatography Measurement of Kynurenine and Kynurenic Acid: Relating Biochemistry to Cognition and Sleep in Rats

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite kynurenic acid (KYNA), an antagonist at both N-methyl-d-aspartate (NMDA) and &#945;7 nicotinic acetylcholine (&#945;7nACh) receptors, has been implicated in cognitive processes in health and disease. As KYNA levels are elevated in the brains of patients with schizophrenia, a malfunction at the glutamatergic and cholinergic receptors is believed to be causally related to cognitive dysfunction, a core domain of the psychopathology of the illness. KYNA may play a pathophysiologically significant role in individuals with schizophrenia. It is possible to elevate endogenous KYNA in the rodent brain by treating animals with the direct bioprecursor kynurenine, and preclinical studies in rats have demonstrated that acute elevations in KYNA may impact their learning and memory processes. The current protocol describes this experimental approach in detail and combines a) a biochemical analysis of blood kynurenine levels and brain KYNA formation (using high-performance liquid chromatography), b) behavioral testing to probe the hippocampal-dependent contextual memory (passive avoidance paradigm), and c) an assessment of sleep-wake behavior [telemetric recordings combining electroencephalogram (EEG) and electromyogram (EMG) signals] in rats. Taken together, a relationship between elevated KYNA, sleep, and cognition is studied, and this protocol describes in detail an experimental approach to understanding function outcomes of kynurenine elevation and KYNA formation in vivo in rats. Results obtained through variations of this protocol will test the hypothesis that the KP and KYNA serve pivotal roles in modulating sleep and cognition in health and disease states.

Alterations in the kynurenine pathway (KP) neuroactive metabolites are implicated in psychiatric illnesses. Investigating the functional outcomes of an altered kynurenine pathway metabolism in vivo in rodents may help elucidate novel therapeutic approaches. The current protocol combines biochemical and behavioral approaches to investigate the impact of an acute kynurenine challenge in rats.

高效液相色谱法测定犬尿氨和 Kynurenic 酸: 对大鼠认知和睡眠的相关生物化学研究

The kynurenine pathway (KP) of tryptophan degradation has been implicated in psychiatric disorders. Specifically, the astrocyte-derived metabolite ...

行为学

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 ...

Integrated Cell Manipulation Platform Coupled with the Single-probe for Mass Spectrometry Analysis of Drugs and Metabolites in Single Suspension Cells

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. The single-probe, a microscale sampling and ionization device, can be coupled with a mass spectrometer for on-line, rapid SCMS analysis of cellular constituents under ambient conditions. Previously, the single-probe SCMS technique was primarily used to measure cells immobilized onto a substrate, limiting the types of cells for studies. In the current study, the single-probe SCMS technology has been integrated with a cell manipulation system, typically used for in vitro fertilization. This integrated cell manipulation and analysis platform uses a cell-selection probe to capture identified individual floating cells and transfer the cells to the single-probe tip for microscale lysis, followed by immediate mass spectrometry analysis. This capture and transfer process removes the cells from the surrounding solution prior to analysis, minimizing the introduction of matrix molecules in the mass spectrometry analysis. This integrated setup is capable of SCMS analysis of targeted patient-isolated cells present in body fluids samples (e.g., urine, blood, saliva, etc.), allowing for potential applications of SCMS analysis to human medicine and disease biology.

An integrated cell manipulation platform is developed for use in conjunction with a single-probe mass spectrometry setup for the on-line analysis of individual suspension cells under ambient conditions.

集成细胞操作平台与单悬架细胞中药物和代谢物质谱分析的单探针相结合

Single cell mass spectrometry (SCMS) enables sensitive detection and accurate analysis of broad ranges of cellular species on the individual-cell level. ...

Quantitative Mass Spectrometric Profiling of Cancer-cell Proteomes Derived From Liquid and Solid Tumors

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic biomarkers. However, methods for quantitative proteomic characterization of patient-derived tumors and in particular their cellular subpopulations are largely lacking. Here we describe an experimental set-up that allows quantitative analysis of proteomes of cancer cell subpopulations derived from either liquid or solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry followed by bioinformatic data analysis. To enrich specific cellular subsets, liquid tumors are first immunophenotyped by flow cytometry followed by FACS-sorting; for solid tumors, laser-capture microdissection is used to purify specific cellular subpopulations. In a second step, proteins are extracted from the purified cells and subsequently combined with a tumor-specific, SILAC-labeled spike-in standard that enables protein quantification. The resulting protein mixture is subjected to either gel electrophoresis or Filter Aided Sample Preparation (FASP) followed by tryptic digestion. Finally, tryptic peptides are analyzed using a hybrid quadrupole-orbitrap mass spectrometer, and the data obtained are processed with bioinformatic software suites including MaxQuant. By means of the workflow presented here, up to 8,000 proteins can be identified and quantified in patient-derived samples, and the resulting protein expression profiles can be compared among patients to identify diagnostic proteomic signatures or potential drug targets.

In-depth analyses of cancer cell proteomes facilitate identification of novel drug targets and diagnostic biomarkers. We describe an experimental workflow for quantitative analysis of (phospho-)proteomes in cancer cell subpopulations derived from liquid and solid tumors. This is achieved by combining cellular enrichment strategies with quantitative Super-SILAC-based mass spectrometry.

对源自液体和固体肿瘤癌症细胞定量蛋白质组质谱剖析

In-depth analyses of cancer cell proteomes are needed to elucidate oncogenic pathomechanisms, as well as to identify potential drug targets and diagnostic ...

医学

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

基于莱萨特的无细胞系统中的代谢物分析液色谱耦合折射指数或质谱检测

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...

Extracellular Purine Metabolism: An HPLC-based Assay to Measure Adenosine Generation and Metabolism in Leukemic Cells

Source: Serra S et al. <a href="https://jove.unicartagenaproxy.elogim.com/t/54124">HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells.</a> J. Vis. Exp. (2016)
This video explains a quantitative reverse phase high-performance liquid chromatography (RP-HPLC) method to measure metabolism of adenosine and associated purines generated by consumption of AMP in extracellular environment of leukemic cancer cells. The protocol describes a sensitive, specific, reliable and reproducible RP-HPLC assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions.

细胞外嘌呤代谢:一种基于 HPLC 的测定法，用于测量白血病细胞中腺苷的产生和代谢

Source: Serra S et al. HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells. J. Vis. ...