Rational design of ¹³C-labeling experiments for metabolic flux analysis in mammalian cells.
Rational design of ¹³C-labeling experiments for metabolic flux analysis in mammalian cells.
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DOI：
10.1186/1752-0509-6-43
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发表时间：
2012-05-16
影响因子：
--
通讯作者：
Antoniewicz MR
中科院分区：
文献类型：
--
作者：
Crown SB;Ahn WS;Antoniewicz MR
13C-代谢通量分析 (13C-MFA) 是探测细胞代谢和阐明体内代谢通量的标准技术。 13C-示踪剂选择是进行 13C-MFA 的重要步骤，然而，当前的方法仅限于试错法，通常侧重于示踪剂设计空间的任意子集。为了系统地探索完整的示踪剂设计空间，特别是对于哺乳动物细胞等复杂系统，迫切需要新的合理方法来识别最佳示踪剂。最近，我们引入了一种基于基本代谢物单元 (EMU) 分解的最佳 13C 示踪剂设计的新框架，其中测量的代谢物被分解为所谓的 EMU 基本向量的线性组合。在这篇文章中，我们将 EMU 方法应用于哺乳动物代谢的现实网络模型，以乳酸作为测量的代谢物。该方法用于选择系统中两种游离通量的最佳示踪剂，即氧化戊糖磷酸途径（oxPPP）通量和丙酮酸羧化酶（PC）的回补。我们的方法基于 EMU 基向量系数相对于自由通量的敏感性分析。通过系数灵敏度的有效分组，导出了简单的示踪剂选择规则，用于哺乳动物网络模型中通量的高分辨率量化。该方法显着减少了可能的示踪剂的数量，并使用数值模拟评估了可行的示踪剂。确定了两种最佳的新型示踪剂，这些示踪剂以前没有被考虑用于哺乳动物细胞的 13C-MFA，特别是用于阐明 oxPPP 通量的 [2,3,4,5,6-13C] 葡萄糖和用于阐明 PC 通量的 [3,4-13C] 葡萄糖。我们证明，与最佳葡萄糖示踪剂相比，13C-谷氨酰胺示踪剂在该系统中表现较差。在这项工作中，我们证明了最佳示踪剂设计不需要是纯粹基于模拟的试错过程；相反，通过应用 EMU 基本向量方法可以获得对示踪剂设计的合理见解。使用这种方法，可以先建立合理的标记规则，以指导选择最佳的 13C 示踪剂，以在复杂的代谢网络模型中进行高分辨率通量阐明。
13C-Metabolic flux analysis (13C-MFA) is a standard technique to probe cellular metabolism and elucidate in vivo metabolic fluxes. 13C-Tracer selection is an important step in conducting 13C-MFA, however, current methods are restricted to trial-and-error approaches, which commonly focus on an arbitrary subset of the tracer design space. To systematically probe the complete tracer design space, especially for complex systems such as mammalian cells, there is a pressing need for new rational approaches to identify optimal tracers. Recently, we introduced a new framework for optimal 13C-tracer design based on elementary metabolite units (EMU) decomposition, in which a measured metabolite is decomposed into a linear combination of so-called EMU basis vectors. In this contribution, we applied the EMU method to a realistic network model of mammalian metabolism with lactate as the measured metabolite. The method was used to select optimal tracers for two free fluxes in the system, the oxidative pentose phosphate pathway (oxPPP) flux and anaplerosis by pyruvate carboxylase (PC). Our approach was based on sensitivity analysis of EMU basis vector coefficients with respect to free fluxes. Through efficient grouping of coefficient sensitivities, simple tracer selection rules were derived for high-resolution quantification of the fluxes in the mammalian network model. The approach resulted in a significant reduction of the number of possible tracers and the feasible tracers were evaluated using numerical simulations. Two optimal, novel tracers were identified that have not been previously considered for 13C-MFA of mammalian cells, specifically [2,3,4,5,6-13C]glucose for elucidating oxPPP flux and [3,4-13C]glucose for elucidating PC flux. We demonstrate that 13C-glutamine tracers perform poorly in this system in comparison to the optimal glucose tracers. In this work, we have demonstrated that optimal tracer design does not need to be a pure simulation-based trial-and-error process; rather, rational insights into tracer design can be gained through the application of the EMU basis vector methodology. Using this approach, rational labeling rules can be established a priori to guide the selection of optimal 13C-tracers for high-resolution flux elucidation in complex metabolic network models.
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影响因子：
8.4
作者：
Antoniewicz, Maciek R.;Kraynie, David F.;Stephanopoulos, Gregory
通讯作者：
Stephanopoulos, Gregory
影响因子：
8.4
作者：
Choi, Jungik;Antoniewicz, Maciek R.
通讯作者：
Antoniewicz, Maciek R.
影响因子：
2.9
作者：
Jeffrey, FMH;Roach, JS;Malloy, CR
通讯作者：
Malloy, CR
DOI：
10.1097/00075197-199809000-00016
发表时间：
1998-09-01
影响因子：
3.1
作者：
Previs, S F;Brunengraber, H
通讯作者：
Brunengraber, H
影响因子：
29
作者：
Burgess, Shawn C.;He, TianTeng;Magnuson, Mark A.
通讯作者：
Magnuson, Mark A.