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Journal article
Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture
Analytical biochemistry, v 474, pp 59-65
01 Apr 2015
PMID: 25572876
Featured in Collection : UN Sustainable Development Goals @ Drexel
Abstract
Acyl-coenzyme A (CoA) thioesters are key metabolites in numerous anabolic and catabolic pathways, including fatty acid biosynthesis and beta-oxidation, the Krebs cycle, and cholesterol and isoprenoid biosynthesis. Stable isotope dilution-based methodology is the "gold standard" for quantitative analyses by mass spectrometry. However, chemical synthesis of families of stable isotope-labeled metabolites such as acyl-CoA thioesters is impractical. Previously, we biosynthetically generated a library of stable isotope internal standard analogs of acyl-CoA thioesters by exploiting the essential requirement in mammals and insects for pantothenic acid (vitamin B5) as a metabolic precursor for the CoA backbone. By replacing pantothenic acid in the cell medium with commercially available [(C3N1)-C-13-N-15]-pantothenic acid, mammalian cells exclusively incorporated [(C3N1)-C-13-N-15]-pantothenate into the biosynthesis of acyl-CoA and acyl-CoA thioesters. We have now developed a much more efficient method for generating stable isotope-labeled CoA and acyl-CoAs from [(C3N1)-C-13-N-15]-pantothenate using stable isotope labeling by essential nutrients in cell culture (SILEC) in Pan6-deficient yeast cells. Efficiency and consistency of labeling were also increased, likely due to the stringently defined and reproducible conditions used for yeast culture. The yeast SILEC method greatly enhances the ease of use and accessibility of labeled CoA thioesters and also provides proof of concept for generating other labeled metabolites in yeast mutants. (C) 2015 Elsevier Inc. All rights reserved.
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Details
- Title
- Production of stable isotope-labeled acyl-coenzyme A thioesters by yeast stable isotope labeling by essential nutrients in cell culture
- Creators
- Nathaniel W. Snyder - Drexel UniversityGregory Tombline - University of RochesterAndrew J. Worth - Translational TherapeuticsRobert C. Parry - Translational TherapeuticsJacob A. Silvers - Translational TherapeuticsKevin P. Gillespie - Translational TherapeuticsSankha S. Basu - Translational TherapeuticsJonathan Millen - University of RochesterDavid S. Goldfarb - University of RochesterIan A. Blair - Translational Therapeutics
- Publication Details
- Analytical biochemistry, v 474, pp 59-65
- Publisher
- Elsevier
- Number of pages
- 7
- Grant note
- T32GM008076 / NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of General Medical Sciences (NIGMS) P30ES013508 / NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA; NIH National Institute of Environmental Health Sciences (NIEHS) P42ES023720; P30ES013508; T32ES019851 / National Institutes of Health; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA Navitor Pharmaceuticals
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- A.J. Drexel Autism Institute
- Web of Science ID
- WOS:000352170800010
- Scopus ID
- 2-s2.0-84923380555
- Other Identifier
- 991019168111604721
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- Collaboration types
- Domestic collaboration
- Web of Science research areas
- Biochemical Research Methods
- Biochemistry & Molecular Biology
- Chemistry, Analytical