Protocol to quantitatively assess glycolysis and related carbon metabolic fluxes using stable isotope tracing in Crabtree-positive yeasts
Summary: Crabtree-positive yeasts rapidly consume glucose via glycolysis, making it difficult to experimentally estimate their actual glycolytic rate or flux. We present a stable isotope labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protocol to quantitatively estimate...
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| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
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| Series: | STAR Protocols |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666166725001923 |
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| Summary: | Summary: Crabtree-positive yeasts rapidly consume glucose via glycolysis, making it difficult to experimentally estimate their actual glycolytic rate or flux. We present a stable isotope labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based protocol to quantitatively estimate glycolytic and related carbon metabolic fluxes using Saccharomyces cerevisiae. This approach defines time windows to capture glucose metabolic intermediate production before label saturation, enabling a comparison of glycolytic flux changes across different cells. This protocol provides a reliable, quantitative approach to study dynamic metabolic fluxes in these cells.For complete details on the use and execution of this protocol, please refer to Vengayil et al., 2024.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. |
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| ISSN: | 2666-1667 |