{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,12]],"date-time":"2025-10-12T04:19:15Z","timestamp":1760242755621,"version":"build-2065373602"},"reference-count":23,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2016,4,23]],"date-time":"2016-04-23T00:00:00Z","timestamp":1461369600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100013886","name":"BE-BASIC Foundation","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100013886","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100013704","name":"CONACyT","doi-asserted-by":"publisher","award":["212059"],"award-info":[{"award-number":["212059"]}],"id":[{"id":"10.13039\/501100013704","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Metabolites"],"abstract":"<jats:p>Ammonium (NH4+) is the most common N-source for yeast fermentations, and N-limitation is frequently applied to reduce growth and increase product yields. While there is significant molecular knowledge on NH4+ transport and assimilation, there have been few attempts to measure the in vivo concentration of this metabolite. In this article, we present a sensitive and accurate analytical method to quantify the in vivo intracellular ammonium concentration in Saccharomyces cerevisiae based on standard rapid sampling and metabolomics techniques. The method validation experiments required the development of a proper sample processing protocol to minimize ammonium production\/consumption during biomass extraction by assessing the impact of amino acid degradation\u2014an element that is often overlooked. The resulting cold chloroform metabolite extraction method, together with quantification using ultra high performance liquid chromatography-isotope dilution mass spectrometry (UHPLC-IDMS), was not only more sensitive than most of the existing methods but also more accurate than methods that use electrodes, enzymatic reactions, or boiling water or boiling ethanol biomass extraction because it minimized ammonium consumption\/production during sampling processing and interference from other metabolites in the quantification of intracellular ammonium. Finally, our validation experiments showed that other metabolites such as pyruvate or 2-oxoglutarate (\u03b1KG) need to be extracted with cold chloroform to avoid measurements being biased by the degradation of other metabolites (e.g., amino acids).<\/jats:p>","DOI":"10.3390\/metabo6020012","type":"journal-article","created":{"date-parts":[[2016,4,25]],"date-time":"2016-04-25T10:02:36Z","timestamp":1461578556000},"page":"12","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Accurate Measurement of the in vivo Ammonium Concentration in Saccharomyces cerevisiae"],"prefix":"10.3390","volume":"6","author":[{"given":"Hugo","family":"Cueto-Rojas","sequence":"first","affiliation":[{"name":"Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Reza","family":"Maleki Seifar","sequence":"additional","affiliation":[{"name":"Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angela","family":"Ten Pierick","sequence":"additional","affiliation":[{"name":"Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sef","family":"Heijnen","sequence":"additional","affiliation":[{"name":"Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aljoscha","family":"Wahl","sequence":"additional","affiliation":[{"name":"Cell Systems Engineering Group, Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2016,4,23]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"616","DOI":"10.1038\/msb.2012.46","article-title":"Need-based activation of ammonium uptake in escherichia coli","volume":"8","author":"Kim","year":"2012","journal-title":"Mol. 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