Real-time cellular exometabolome analysis with a microfluidic-mass spectrometry platform.
Real-time cellular exometabolome analysis with a microfluidic-mass spectrometry platform.
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DOI：
10.1371/journal.pone.0117685
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Wikswo JP
中科院分区：
文献类型：
--
作者：
Marasco CC;Enders JR;Seale KT;McLean JA;Wikswo JP
为了解决跟踪众多的信号分子和代谢物，这是生物复杂性的基础上的挑战，我们描述了一种策略，以扩大动态系统生物学的分析技术。利用微流体、在线质谱和质谱技术，我们构建并验证了一个非常适合以高时间分辨率对细胞微环境进行采样的平台。我们的平台在以下方面取得了成功：自动化细胞刺激和微环境控制;由于表面钝化而减少了对聚二甲基硅氧烷的非特异性吸附;实时在线样品收集;近实时样品制备以去除盐;以及实时在线质谱。当与“培养”实验结合超高效液相色谱-电喷雾电离-离子迁移-质谱的基准相比时，（UPLC-ESI-IM-MS），我们的平台解决了自分泌和旁分泌信号稀释引起的体积挑战问题，并大大减少了样品制备和数据收集时间，同时减少来自操作细胞和培养基的各种手动方法的不希望的外部影响（例如，细胞离心）。为了在生物学上验证这个系统，我们专注于Jurkat T细胞对微环境刺激的细胞反应。这些刺激的应用，结合细胞的代谢过程，导致营养素的消耗和生物分子（统称为外代谢组）的分泌的变化，这使得与其他细胞或组织的通信和废物的消除成为可能。可卡因的幼稚和经验丰富的T细胞代谢被用作示例性系统，以确认平台的能力，突出其代谢物发现应用的潜力，并探索T细胞药物暴露的免疫记忆。我们的平台被证明能够检测幼稚和有经验的Jurkat T细胞之间的代谢组学变化，并突出了外代谢组随时间的动态变化。可卡因代谢产物苯甲酰爱子碱的上调，在有经验的T细胞中被注意到，表明可卡因暴露的潜在细胞记忆。这些代谢组学差异在类似的传统“培养中”UPLC-ESI-IM-MS实验中不存在，进一步证明了该平台的能力。
To address the challenges of tracking the multitude of signaling molecules and metabolites that is the basis of biological complexity, we describe a strategy to expand the analytical techniques for dynamic systems biology. Using microfluidics, online desalting, and mass spectrometry technologies, we constructed and validated a platform well suited for sampling the cellular microenvironment with high temporal resolution. Our platform achieves success in: automated cellular stimulation and microenvironment control; reduced non-specific adsorption to polydimethylsiloxane due to surface passivation; real-time online sample collection; near real-time sample preparation for salt removal; and real-time online mass spectrometry. When compared against the benchmark of “in-culture” experiments combined with ultraperformance liquid chromatography-electrospray ionization-ion mobility-mass spectrometry (UPLC-ESI-IM-MS), our platform alleviates the volume challenge issues caused by dilution of autocrine and paracrine signaling and dramatically reduces sample preparation and data collection time, while reducing undesirable external influence from various manual methods of manipulating cells and media (e.g., cell centrifugation). To validate this system biologically, we focused on cellular responses of Jurkat T cells to microenvironmental stimuli. Application of these stimuli, in conjunction with the cell’s metabolic processes, results in changes in consumption of nutrients and secretion of biomolecules (collectively, the exometabolome), which enable communication with other cells or tissues and elimination of waste. Naïve and experienced T-cell metabolism of cocaine is used as an exemplary system to confirm the platform’s capability, highlight its potential for metabolite discovery applications, and explore immunological memory of T-cell drug exposure. Our platform proved capable of detecting metabolomic variations between naïve and experienced Jurkat T cells and highlights the dynamics of the exometabolome over time. Upregulation of the cocaine metabolite, benzoylecgonine, was noted in experienced T cells, indicating potential cellular memory of cocaine exposure. These metabolomics distinctions were absent from the analogous, traditional “in-culture” UPLC-ESI-IM-MS experiment, further demonstrating this platform’s capabilities.
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影响因子：
4.2
作者：
Chao, Chun;Jacobson, Lisa P.;Detels, Roger
通讯作者：
Detels, Roger
影响因子：
2.3
作者：
Enders JR;Marasco CC;Kole A;Nguyen B;Sevugarajan S;Seale KT;Wikswo JP;McLean JA
通讯作者：
McLean JA
影响因子：
6.1
作者：
Darby SG;Moore MR;Friedlander TA;Schaffer DK;Reiserer RS;Wikswo JP;Seale KT
通讯作者：
Seale KT
影响因子：
1.9
作者：
Shotwell MS;Drake KJ;Sidorov VY;Wikswo JP
通讯作者：
Wikswo JP
影响因子：
6.1
作者：
Faley, Shannon L.;Copland, Mhairi;Cooper, Jonathan M.
通讯作者：
Cooper, Jonathan M.