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Nat Protoc. 2019 Feb;14(2):313-330. doi: 10.1038/s41596-018-0102-x.

Ex vivo and in vivo stable isotope labelling of central carbon metabolism and related pathways with analysis by LC-MS/MS.

Author information

1
Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA.
2
Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Boston, MA, USA.
3
Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA.
4
Graduate Program in Chemical Biology, University of Michigan, Ann Arbor, MI, USA.
5
Department of Medicine, Harvard Medical School, Boston, MA, USA.
6
Department of Biochemistry and Molecular Genetics, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA.
7
Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA, USA.
8
Rogel Cancer Center, University of Michigan, Ann Arbor, MI, USA. clyssiot@med.umich.edu.
9
Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA. clyssiot@med.umich.edu.
10
Department of Internal Medicine, Division of Gastroenterology, University of Michigan, Ann Arbor, MI, USA. clyssiot@med.umich.edu.
11
Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA. jasara@bidmc.harvard.edu.
12
Mass Spectrometry Core, Beth Israel Deaconess Medical Center, Boston, MA, USA. jasara@bidmc.harvard.edu.
13
Department of Medicine, Harvard Medical School, Boston, MA, USA. jasara@bidmc.harvard.edu.

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.

PMID:
30683937
DOI:
10.1038/s41596-018-0102-x
[Indexed for MEDLINE]

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