An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in E. coli

Emily J Geddes, Zhong Li, Paul J Hergenrother

Research output: Contribution to journalReview articlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)4833-4854
Number of pages22
JournalNature Protocols
Issue number10
Early online dateSep 3 2021
StatePublished - Oct 2021

ASJC Scopus subject areas

  • General Biochemistry, Genetics and Molecular Biology


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