Journal
NATURE PROTOCOLS
Volume 14, Issue 2, Pages 313-330Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-018-0102-x
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Funding
- National Institutes of Health [5P01CA120964, 5R35CA197459, 5P30CA006516]
- BIDMC Research Capital Fund
- Pancreatic Cancer Action Network/AACR Pathway to Leadership award [13-70-25-LYSS]
- Dale F. Frey Award for Breakthrough Scientists from the Damon Runyon Cancer Research Foundation [DFS-09-14]
- V Foundation for Cancer Research [V2016-009]
- Kimmel Scholar Award from the Sidney Kimmel Foundation for Cancer Research [SKF-16-005]
- 2017 AACR NextGen Grant for Transformative Cancer Research [17-20-01-LYSS]
- University of Michigan Program in Chemical Biology Graduate Assistance in Areas of National Need (GAANN) award
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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 (C-13) and nitrogen-15 (N-15) 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 C-13-labeled glucose, C-13-glutamine and N-15-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 C-13 or N-15 incorporation. The entire procedure takes similar to 6-7 h for a single sample from experimental labelling and metabolite extraction to peak integration.
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