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.

Metabolic reprogramming of acute lymphoblastic leukemia cells in response to glucocorticoid treatment

“…For the metabolomics and isotope-tracing experiments, a custom-synthesized RPMI medium was used according to standard formulation (Sigma), using the following isotopomers: 70% 1- 13 C-glucose, 50% U- 13 C, 15 N-glutamine, 60% 3- 13 C-serine, and 50% U- 13 C-methionine. For metabolite extraction, cells were washed twice with Hank’s balanced salt solution, and the resulting pellet was extracted with cold HPLC-grade methanol and analyzed using a Thermo QExactive orbitrap instrument coupled to a HILIC chromatography system, as previously described 49 . The identity of metabolites were confirmed by matching retention times against those of pure standards, and additionally supported by comparing the observed mass isotopomers to those expected given the isotope tracers used.…”

Noninvasive rapid detection of metabolic adaptation in activated human T lymphocytes by hyperpolarized 13C magnetic resonance

“…www.nature.com/scientificreports www.nature.com/scientificreports/ based on accurate mass and retention time matching against an in-house database containing data on 600 polar metabolite standards (analyzed in the same analytical conditions). The Extracted Ion Chromatogram areas (EICs) of each isotopologue (M + , M + 1, M + 2 and M + 3) were corrected for natural isotope abundance 37 and the label incorporation or 13 C enrichment of lactate was calculated based on relative isotopologue abundance (in %), in each one of two analyzed conditions 38 .…”

Sorting cells alters their redox state and cellular metabolome

“…An experimental strategy that is commonly employed to purify populations of cells from complex samples is fluorescence-activated cell sorting or FACS [11] . One potential workflow is to isolate specific types of cells from complex samples by FACS and subsequently quench their metabolism prior to extracting metabolites for mass spectrometry analysis [12] . While it is provocative to imagine stopping metabolism by enzyme inactivation prior to cell sorting, conventional methods for quenching metabolism are not compatible with FACS.…”