Abstract
Targeted tandem mass spectrometry (LC–MS/MS) has been extremely useful for profiling small molecules extracted from biological sources, such as cells, bodily fluids and tissues. Here, we present a protocol for analysing incorporation of the non-radioactive stable isotopes carbon-13 (13C) and nitrogen-15 (15N) into polar metabolites in central carbon metabolism and related pathways. Our platform utilizes selected reaction monitoring (SRM) with polarity switching and amide hydrophilic interaction liquid chromatography (HILIC) to capture transitions for carbon and nitrogen incorporation into selected metabolites using a hybrid triple quadrupole (QQQ) mass spectrometer. This protocol represents an extension of a previously published protocol for targeted metabolomics of unlabeled species and has been used extensively in tracing the metabolism of nutrients such as 13C-labeled glucose, 13C-glutamine and 15N-glutamine in a variety of biological settings (e.g., cell culture experiments and in vivo mouse labelling via i.p. injection). SRM signals are integrated to produce an array of peak areas for each labelling form that serve as the output for further analysis. The processed data are then used to obtain the degree and distribution of labelling of the targeted molecules (termed fluxomics). Each method can be customized on the basis of known unlabeled Q1/Q3 SRM transitions and adjusted to account for the corresponding 13C or 15N incorporation. The entire procedure takes ~6–7 h for a single sample from experimental labelling and metabolite extraction to peak integration.
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Acknowledgements
This research was supported by grants from the National Institutes of Health (5P01CA120964 to J.M.A. and B.D.M.; 5R35CA197459 to B.D.M. and J.M.A.; and 5P30CA006516 to J.M.A.) and the BIDMC Research Capital Fund for funding the mass spectrometry instrumentation. C.A.L. was supported by a Pancreatic Cancer Action Network/AACR Pathway to Leadership award (13-70-25-LYSS), a Dale F. Frey Award for Breakthrough Scientists from the Damon Runyon Cancer Research Foundation (DFS-09-14), a Junior Scholar Award from The V Foundation for Cancer Research (V2016-009), a Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research (SKF-16-005), and a 2017 AACR NextGen Grant for Transformative Cancer Research (17-20-01-LYSS). D.M.K was supported by a University of Michigan Program in Chemical Biology Graduate Assistance in Areas of National Need (GAANN) award. We thank L.C. Cantley (Weill-Cornell MC) and G.M. Wulf (BIDMC/HMS) for helpful discussions and the donation of K14 cells.
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Authors and Affiliations
Contributions
J.M.A. developed the platform, incorporated the methods, analysed data and wrote the protocol. C.A.L. and D.M.K. helped to develop the SRM transitions for labeled metabolites, generated samples for testing the protocol, analysed data and edited the protocol. B.D.M. and I.B.S. helped to generate the SRM transitions for labeled metabolites, generated samples for testing the protocol and edited the protocol. M.Y. helped to incorporate methods, maintained the platform and helped with sample generation and data analysis. H.H. helped to develop the SRM transitions for labeled metabolites, generated samples for testing the protocol and helped with data analysis. S.B.B. incorporated methods and generated labeled samples for testing the protocol.
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Protocol to which this paper is an extension
Yuan, M., Breitkopf, S. B., Yang, X. & Asara, J. M. Nat. Protoc. 7, 872–881 (2012) https://www.nature.com/articles/nprot.2012.024
Key references using this protocol
Son, J. et al. Nature 496, 101–105 (2013): http://www.nature.com/articles/nature12040
Ben-Sahra, I., Howell, J. J., Asara, J. M. & Manning, B. D. Science 339, 1323–1328 (2013): http://science.sciencemag.org/content/339/6125/1323
Ying, H. et al. Cell 149, 656–670 (2012): https://www.cell.com/cell/abstract/S0092-8674(12)00352-2
Ben-Sahra, I., Hoxhaj, G., Ricoult, S. J. H., Asara, J. M. & Manning, B. D.: Science 351, 728–733 (2016) http://science.sciencemag.org/content/351/6274/728
This protocol is an extension to: Yuan, M. et al., Nat. Protoc. 7, 872–881 (2012), doi:10.1038/nprot.2012.024; published online 12 April 2012.
Supplementary information
Supplementary Dataset 1
Supplementary Dataset 1
Supplementary Dataset 2
Supplementary Dataset 2
Supplementary Dataset 3
Supplementary Dataset 3
Supplementary Dataset 4
Supplementary Dataset 4
Supplementary Dataset 5
Supplementary Dataset 5
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Yuan, M., Kremer, D.M., Huang, H. et al. Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC–MS/MS. Nat Protoc 14, 313–330 (2019). https://doi.org/10.1038/s41596-018-0102-x
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DOI: https://doi.org/10.1038/s41596-018-0102-x
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