A Protocol for Lab‐Scale Production of 13 C‐Labeled Yeast Extract as Internal Standard for Metabolomics and Quantification of Intracellular Metabolites
Miriam Cammaert, Rosalie Wouters, Jitske van Ede, Erik de Hulster, Christiaan Mooiman, Patricia van Dam, Martin Pabst, Walter van Gulik, Pascale Daran‐LapujadeAbstract
Metabolomics enables the profiling of small‐molecule metabolites, thereby capturing the biochemical state of a living organism at a given moment and making it possible to monitor the organism's cellular responses to stimuli. This technique has become a powerful tool in pharmaceutical research, the food industry, and microbial research. Metabolomics aims to obtain an unbiased metabolic profile; however, this is complicated by compound instability, the requirement for complex and often extensive sample processing, and nonlinear responses in mass spectrometry. Therefore, correcting for metabolite loss and mass‐spectrometry‐related artifacts is essential; this is typically achieved through relative quantification against an isotopically labeled internal standard for each metabolite of interest. This article describes a method to produce 13 C‐labeled yeast extract and its use as internal standard for metabolomics. More specifically, it provides step‐by‐step protocols for the fed‐batch fermentation, quenching, metabolite extraction, and LC‐MS and GC‐MS characterization of the internal standard. It also includes a protocol explaining how to use the internal standard for the quantification of metabolites in yeast samples. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC.
Basic Protocol 1 : Trial run—fed‐batch fermentation
Support Protocol 1 : Dry weight determination
Basic Protocol 2 : Quenching and washing of the cell samples and metabolite extraction
Basic Protocol 3 : Identification of intracellular metabolites using LC‐MS
Basic Protocol 4 : Identification of intracellular metabolites using GC‐MS
Basic Protocol 5 : Real run—fed‐batch fermentation
Basic Protocol 6 : LC‐MS characterization of the internal standard
Basic Protocol 7 : GC‐MS characterization of the internal standard
Basic Protocol 8 : Quantification of metabolites in yeast samples