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SILEC: a protocol for generating and using isotopically labeled coenzyme A mass spectrometry standards

Nature Protocols volume 7, pages 1–11 (2012)Cite this article

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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Figure 1: LC-MS analysis of acyl-CoA thioesters.
Figure 2
Figure 3: Pantothenate and coenzyme A biosynthesis.
Figure 4: General scheme for stable isotope labeling by essential nutrients in cell culture (SILEC).
Figure 5: SILEC labeling and 'customization' of CoA metabolome.
Figure 6: Biosynthetic generation of isotopically labeled menadione-CoA.
Figure 7: LC-SRM/MS chromatograms of CoASH harvested from [13C315N]-pantothenate-labeled cells.

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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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Authors and Affiliations

  1. Centers of Excellence in Environmental Toxicology and Cancer Pharmacology, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Sankha S Basu & Ian A Blair

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  1. Sankha S Basu
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Both authors wrote the paper and designed the experiments. S.S.B. conducted the actual experiments.

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Correspondence to Ian A Blair.

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Supplementary information

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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