Abstract
Stable isotopic tracer analysis is a technique used to determine carbon or nitrogen atom incorporation into biological systems. A number of mass spectrometry based approaches have been developed for this purpose, including high-resolution tandem mass spectrometry (HR-LC-MS/MS), selected reaction monitoring (SRM) and parallel reaction monitoring (PRM). We have developed an approach for analyzing untargeted metabolomic and lipidomic datasets using high-resolution mass spectrometry with polarity switching and implemented our approach in the open-source R script IsoSearch and in Scaffold Elements software. Using our strategy, which requires an unlabeled reference dataset and isotope labeled datasets across various biological conditions, we traced metabolic isotopomer alterations in breast cancer cells (MCF-7) treated with the metabolic drugs 2-deoxy-glucose, 6-aminonicotinamide, compound 968, and rapamycin. Metabolites and lipids were first identified by the commercial software Scaffold Elements and LipidSearch, then IsoSearch successfully profiled the 13C-isotopomers extracted metabolites and lipids from 13C-glucose labeled MCF-7 cells. The results interpreted known models, such as glycolysis and pentose phosphate pathway inhibition, but also helped to discover new metabolic/lipid flux patterns, including a reactive oxygen species (ROS) defense mechanism induced by 6AN and triglyceride accumulation in rapamycin treated cells. The results suggest the IsoSearch/Scaffold Elements platform is effective for studying metabolic tracer analysis in diseases, drug metabolism, and metabolic engineering for both polar metabolites and non-polar lipids.
Highlights
Metabolic flux analysis (MFA) using stable isotope tracers is a technique used to investigate the intracellular metabolic rate of cells and organisms [1,2]
As an example of the technology, we show MCF-7 human breast cancer cells after treatment with known inhibitors of central carbon metabolism and compare how universally labeled 13C[6]-glucose carbon atoms are tracked throughout metabolic processes
Isotopic tracer flux analyses typically report the detection of molecules present in well-known metabolic pathways
Summary
Metabolic flux analysis (MFA) using stable isotope tracers is a technique used to investigate the intracellular metabolic rate of cells and organisms [1,2]. Stable isotope labeled nutrients, such as 13C-glucose and 15N-glutamine, are frequently used in targeted mass spectrometry (GC-MS or LC-MS) based approaches to analyze metabolite isotopomer incorporations [1,6,7]. Many laboratories have developed various software tools to interpret the MFA results generated by NMR [14], GC-MS [15,16,17], or LC-MS, such as SUMOFLUX [18], 13C-FLUX [14,19], X13CMS [20], and Omix [21]. The 13C-FLUX results can be visualized by the Omix software Both SUMOFLUX and 13C-FLUX can process targeted metabolic flux results, they are less powerful for managing untargeted fluxomics results and discovering new metabolic flux mechanisms. X13CMS aims to analyze untargeted metabolic isotopic data by requiring a paired isotope labeled experiment with an unlabeled sample, and differentiates the isotopomers based on the mass differences. The dependence on XCMS outputs makes X13CMS unlikely to incorporate results from other software such as Proteome Software’s Scaffold Elements [25] and Thermo’s LipidSearch
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