Metabolite Profiling and Stable Isotope Tracing in Sorted Subpopulations of Mammalian Cells

Abstract

Biological samples such as tissues, blood, or tumors are often complex and harbor heterogeneous populations of cells. Separating out specific cell types or subpopulations from such complex mixtures to study their metabolic phenotypes is challenging because experimental procedures for separation may disturb the metabolic state of cells. To address this issue, we developed a method for analysis of cell subpopulations using stable isotope tracing and fluorescence-activated cell sorting followed by liquid chromatography-high-resolution mass spectrometry. To ensure a faithful representation of cellular metabolism after cell sorting, we benchmarked sorted extraction against direct extraction. While peak areas differed markedly with lower signal for amino acids but higher signal for nucleotides, mass isotopomer distributions from sorted cells were generally in good agreement with those obtained from direct extractions, indicating that they reflect the true metabolic state of cells prior to sorting. In proof-of-principle studies, our method revealed metabolic phenotypes specific to T cell subtypes, and also metabolic features of cells in the committed phase of the cell division cycle. Our approach enables studies of a wide range of adherent and suspension cell subpopulations, which we anticipate will be of broad importance in cell biology and biomedicine.

Figure 1.

Schematic diagram of the experimental design. a) Comparison of sorted cells populations based on isotope label content. The complex sample is pulse labelled prior to sorting, and differences between subpopulation are reflected in the MIDs. Subpopulations extracts are analyzed separately by LC-HRMS. b) Design of validation experiment. Metabolomics of mock sorted cells were benchmarked with dish and pellet extracts.

Figure 2.

Peak area data from the validation experiment. a) Venn diagram of good quality peaks detected in dish, pellet, and sorted extracts, b) Scatter plots of peak areas between dish, pellet and sorted extracts. All replicates are shown and connected by lines, c) Heat map of normalized peak areas, clustered by metabolites and samples. ncl: nucleotides, aa: amino acids. d) Principal component analysis (PCA) of normalized peak areas. PVE: percentage of variance explained. Red: sorted, blue: pellet, black: dish extracted cells. e) CV (Coefficient of Variation) of peak areas of 65 metabolites from plate, pellet and sorted extracts. Metabolites are plotted in increasing order of mean CV of the extracts.

Figure 3.

MID data from the validation experiment. (a and b) Heat map of ¹⁵N (a) ¹³C (b) enrichment clustered by metabolites and samples. Unlabeled metabolites, mainly essential nutrients are not shown. c) ¹³C-¹⁵N MIDs of Glutamate. (d) ¹³C MIDs of Lactate, The error bars are standard deviations of triplicate measurements in (a) and (b). (e) Standard deviation (st.dev) of MI fractions of 60 MIDs. (−) stands for negative mode, (+) stands for positive mode. Descriptions of metabolites can be found in Table S-1.

Figure 4.

Adenosine is differentially labelled in CD8⁺ and CD4⁺ cells. Error bars in bar charts represent standard deviations. Array plots (inset) show MI fractions. ‘x’ stands for missing values, which is noise manually corrected to ‘0’.

Figure 5.

Cytidine is labeled differently in G₁–G₀ and S-G₂-M cells. a) Cytidine ¹³C enrichment in G₁–G₀ and S-G₂-M phases of the cell cycle. Dashed line stands for carbon enrichment from natural isotope. b) Cytidine MIDs shown as array plots in G₁–G₀ and S-G₂-M phases.