An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli.
An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli.
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LC-MS/MS 测定和基于网络的补充工具，用于量化和预测大肠杆菌中的化合物积累。
DOI：
10.1038/s41596-021-00598-y
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发表时间：
2021-10
期刊：
影响因子：
14.8
通讯作者：
Hergenrother PJ
中科院分区：
文献类型：
--
作者：
Geddes EJ;Li Z;Hergenrother PJ
新型广谱抗生素的发现非常困难，这主要是由于革兰氏阴性菌膜的不渗透性，以及对化合物应具有的促进其在细胞内积累的物理化学性质的理解不足。为了应对这一挑战，已经建立了许多用于评估革兰氏阴性菌中细胞内化合物积累的方法，包括经典的基于放射性和荧光的方法。最近开发的利用液相色谱-串联质谱法（LC-MS/MS）的蓄积测定法规避了对标记化合物的要求，从而能够评估范围更广的小分子。我们使用基于LC-MS/MS的测定法对大肠杆菌中的蓄积趋势进行了无偏倚研究，从而制定了eNTRy规则，该规则规定化合物最有可能在大肠杆菌中蓄积。大肠杆菌，如果它具有可电离的氮，具有低的三维性并且相对刚性。为了帮助执行eNTRy规则，我们开发了一个补充的网络工具eNTRyway，它可以计算相关的属性并预测化合物的积累。在这里，我们提供了一个全面的协议，分析和预测细胞内积累的小分子在大肠杆菌。使用基于LC-MS/MS的测定（需要约2天）和eNTRyway（一种易于被任何微生物学、生物化学或化学生物学实验室采用的工作流程）对大肠杆菌进行检测。
Novel classes of broad-spectrum antibiotics have been extremely difficult to discover, largely due to the impermeability of the Gram-negative membranes coupled with a poor understanding of the physicochemical properties a compound should possess to promote its accumulation inside the cell. To address this challenge, numerous methodologies for assessing intracellular compound accumulation in Gram-negative bacteria have been established, including classic radiometric and fluorescence-based methods. The recent development of accumulation assays that utilize liquid chromatography–tandem mass spectrometry (LC-MS/MS) have circumvented the requirement for labeled compounds, enabling assessment of a substantially broader range of small molecules. Our unbiased study of accumulation trends in Escherichia coli using an LC-MS/MS-based assay led to the development of the eNTRy rules, which stipulate that a compound is most likely to accumulate in E. coli if it has an ionizable Nitrogen, has low Three-dimensionality and is relatively Rigid. To aid in the implementation of the eNTRy rules, we developed a complementary web tool, eNTRyway, which calculates relevant properties and predicts compound accumulation. Here we provide a comprehensive protocol for analysis and prediction of intracellular accumulation of small molecules in E. coli using an LC-MS/MS-based assay (which takes ~2 d) and eNTRyway, a workflow that is readily adoptable by any microbiology, biochemistry or chemical biology laboratory.
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影响因子：
5.3
作者：
Perlmutter SJ;Geddes EJ;Drown BS;Motika SE;Lee MR;Hergenrother PJ
通讯作者：
Hergenrother PJ
影响因子：
4
作者：
Davis, Tony D.;Gerry, Christopher J.;Tan, Derek S.
通讯作者：
Tan, Derek S.
影响因子：
2.7
作者：
Li, Yangxiong;Gardner, Jessi J.;Duerfeldt, Adam S.
通讯作者：
Duerfeldt, Adam S.
影响因子：
5.2
作者：
de Cristobal, Ricardo E.;Vincent, Paula A.;Salomon, Raul A.
通讯作者：
Salomon, Raul A.
影响因子：
4.9
作者：
Krishnamoorthy, Ganesh;Wolloscheck, David;Zgurskaya, Helen I.
通讯作者：
Zgurskaya, Helen I.