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Large-Scale Profiling of Cellular Metabolic Activities Using Deep 13C Labeling Medium

  • Nina Grankvist
  • Jeramie D. Watrous
  • Mohit Jain
  • Roland Nilsson
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2088)

Abstract

The recently developed deep labeling method allows for large-scale profiling of metabolic activities in human cells or tissues using isotope tracing with a highly 13C enriched culture medium in combination with liquid chromatography–high resolution mass spectrometry. This method generates mass spectrometry data sets where endogenous cellular products can be identified, and active pathways can be determined from observed 13C mass isotopomers of the various metabolites measured. Here we describe in detail the experimental procedures for deep labeling experiments in cultured mammalian cells, including synthesis of the deep labeling medium, experimental considerations for cell culture, metabolite extractions and sample preparation, and liquid chromatography–mass spectrometry. We also outline a workflow for the downstream data analysis using publicly available software.

Key words

Cell culture Stable isotope tracing experiments Metabolism Metabolomics LC-HRMS 

Notes

Acknowledgments

This work was supported by grants from the Swedish Foundation for Strategic Research (FFL12-0220.006) and Karolinska Institutet to R.N., the National Institutes of Health (NIH) 1R01ES027595 and 1S10OD020025 to M.J., and NIH K01DK116917 to J.D.W.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Nina Grankvist
    • 1
    • 2
    • 3
    • 4
    • 5
  • Jeramie D. Watrous
    • 4
    • 5
  • Mohit Jain
    • 4
    • 5
  • Roland Nilsson
    • 1
    • 2
    • 3
  1. 1.Cardiovascular Medicine Unit, Department of Medicine, SolnaKarolinska InstitutetStockholmSweden
  2. 2.Karolinska University HospitalStockholmSweden
  3. 3.Center for Molecular MedicineKarolinska InstitutetStockholmSweden
  4. 4.Department of MedicineUniversity of California San DiegoLa JollaUSA
  5. 5.Department of PharmacologyUniversity of California San DiegoLa JollaUSA

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