Real-time metabolome profiling of the metabolic switch between starvation and growth

Link, Hannes; Fuhrer, Tobias; Gerosa, Luca; Zamboni, Nicola; Sauer, Uwe

doi:10.1038/nmeth.3584

Cited by 217 publications

(190 citation statements)

References 37 publications

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“…Direct-injection MS has also shown substantial potential for metabolomics analysis of cellular extracts (92–94). Even over the brief injection window, a large number of ions are detected, corresponding to a similar number of primary metabolite masses as in a standard LC-MS run.…”

Section: High-throughput Mass Spectrometrymentioning

confidence: 99%

See 1 more Smart Citation

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

Klein

et al. 2017

Annu. Rev. Biochem.

370

341

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Metabolites are the small biological molecules involved in energy conversion and biosynthesis. Studying metabolism is inherently challenging due to metabolites’ reactivity, structural diversity, and broad concentration range. Herein, we review the common pitfalls encountered in metabolomics and provide concrete guidelines for obtaining accurate metabolite measurements, focusing on water-soluble primary metabolites. We show how seemingly straightforward sample preparation methods can introduce systematic errors (e.g., owing to interconversion among metabolites) and how proper selection of quenching solvent (e.g., acidic acetonitrile:methanol:water) can mitigate such problems. We discuss the specific strengths, pitfalls, and best practices for each common analytical platform: liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), and enzyme assays. Together this information provides a pragmatic knowledge base for carrying out biologically informative metabolite measurements.

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Section: High-throughput Mass Spectrometrymentioning

confidence: 99%

“…Thus, ion suppression is not so severe as to eliminate signals for most metabolites. The improved throughput has enabled some important applications, including screening of whole-genome knockout collections and fine-grained kinetic analysis of the metabolome of bacteria undergoing dynamic perturbations (94). …”

Section: High-throughput Mass Spectrometrymentioning

confidence: 99%

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

Klein

et al. 2017

Annu. Rev. Biochem.

370

341

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“…Thus, studying time-resolved responses of cell cultures to environmental changes or upon the application of a defined compound dosage often requires continuous read-out to ensure that occurrences of important events are not missed 35 . In well-based approaches, liquid handling is discrete, which engenders a high risk of missing events.…”

Section: Introductionmentioning

confidence: 99%

Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks

et al. 2016

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Microfluidics is becoming a technology of growing interest for building microphysiological systems with integrated read-out functionalities. Here we present the integration of enzyme-based multi-analyte biosensors into a multi-tissue culture platform for 'body-on-a-chip' applications. The microfluidic platform is based on the technology of hanging-drop networks, which is designed for the formation, cultivation, and analysis of fluidically interconnected organotypic spherical three-dimensional (3D) microtissues of multiple cell types. The sensor modules were designed as small glass plug-ins featuring four platinum working electrodes, a platinum counter electrode, and an Ag/AgCl reference electrode. They were placed directly into the ceiling substrate from which the hanging drops that host the spheroid cultures are suspended. The electrodes were functionalized with oxidase enzymes to enable continuous monitoring of lactate and glucose through amperometry. The biosensors featured high sensitivities of 322 ± 41 nA mM − 1 mm − 2 for glucose and 443 ± 37 nA mM − 1 mm − 2 for lactate; the corresponding limits of detection were below 10 μM. The proposed technology enabled tissue-size-dependent, real-time detection of lactate secretion from single human colon cancer microtissues cultured in the hanging drops. Furthermore, glucose consumption and lactate secretion were monitored in parallel, and the impact of different culture conditions on the metabolism of cancer microtissues was recorded in real-time.

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“…However, -omics data types are often not utilized in routine screening of new production strain candidates because of high costs and low throughput (Hansen et al, 2017). Of the various -omics data types available, metabolomics provides the greatest potential to gain rich and deep insight on strain physiology at a lower cost and higher throughput (Fuhrer et al, 2011;Guder et al, 2017;Link et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli

McCloskey

Schrübbers

et al. 2018

Metabolic Engineering

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A B S T R A C TFast metabolite quantification methods are required for high throughput screening of microbial strains obtained by combinatorial or evolutionary engineering approaches. In this study, a rapid RIP-LC-MS/MS (RapidRIP) method for high-throughput quantitative metabolomics was developed and validated that was capable of quantifying 102 metabolites from central, amino acid, energy, nucleotide, and cofactor metabolism in less than 5 minutes. The method was shown to have comparable sensitivity and resolving capability as compared to a full length RIP-LC-MS/MS method (FullRIP). The RapidRIP method was used to quantify the metabolome of seven industrial strains of E. coli revealing significant differences in glycolytic, pentose phosphate, TCA cycle, amino acid, and energy and cofactor metabolites were found. These differences translated to statistically and biologically significant differences in thermodynamics of biochemical reactions between strains that could have implications when choosing a host for bioprocessing.

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Real-time metabolome profiling of the metabolic switch between starvation and growth

Cited by 217 publications

References 37 publications

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

Metabolite Measurement: Pitfalls to Avoid and Practices to Follow

Multi-analyte biosensor interface for real-time monitoring of 3D microtissue spheroids in hanging-drop networks

RapidRIP quantifies the intracellular metabolome of 7 industrial strains of E. coli

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