Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis
Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis
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DOI：
10.3389/fmicb.2020.00197
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发表时间：
2020-02-13
影响因子：
5.2
通讯作者：
Hatzenpichler, Roland
中科院分区：
文献类型：
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作者：
Steward, Katherine F.;Eilers, Brian;Hatzenpichler, Roland
BioOrthogonal Non-Canonical Amino Acid Tagging（BONCAT）是一种在群落和整个生物体内的单细胞水平上跟踪蛋白质合成的强大工具。BONCAT的一个基本前提是，用于跟踪翻译活性的非经典氨基酸（NCAA）不会显著改变细胞生理学。如果NCAA会引起细胞代谢状态的变化，那么对BONCAT研究的解释可能具有挑战性。为了解决这一知识缺口，我们使用了一个全球代谢组学分析来评估NCAA掺入的细胞内效应。测试了两种NCAA：L-叠氮基高丙氨酸（AHA）和L-高炔丙基甘氨酸（HPG）; L-甲硫氨酸（MET）用作最小应激基线对照。使用液相色谱-质谱（LC-MS）和核磁共振（NMR）表征大肠杆菌培养物的细胞内代谢产物谱，并使用XCMS和MetaboAnalyst进行多变量统计分析。结果表明，与NCAA兴奋剂诱导代谢的变化，但是，代谢的影响并不显着。在第二组实验中，将培养物置于轻度应力下以模拟真实世界的环境条件，显示出更一致和更稳健的扰动。改变的途径包括氨基酸和蛋白质合成，胆碱和甜菜碱，以及TCA循环。在全球范围内，这些变化在统计学上是微小的，表明NCAA不太可能在掺入过程中对细胞产生重大影响。我们的研究结果是一致的NCAA兴奋剂在充满条件下，并将这些结果扩展到环境相关条件下的细菌生长与以前的报告。我们的工作突出了代谢组学研究在检测细胞对生长条件的反应方面的力量，以及NMR和LCMS作为组学工具的互补性。
BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.