Abstract
Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [13C315N]-pantothenate (vitamin B5), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2–3 weeks.
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Acknowledgements
The support of US National Institutes of Health grants U01ES016004, P30ES013508 and 5T32HL007439 is gratefully acknowledged.
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Both authors wrote the paper and designed the experiments. S.S.B. conducted the actual experiments.
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Supplementary Table 1
Protonated molecule m/z values for endogenous and labeled CoA species. (PPT 153 kb)
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Basu, S., Blair, I. SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards. Nat Protoc 7, 1–11 (2012). https://doi.org/10.1038/nprot.2011.421
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DOI: https://doi.org/10.1038/nprot.2011.421
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