title: 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.
authors: Irena
authors: I
authors: Roci
authors: Hector
authors: H
authors: Gallart-Ayala
authors: Angelika
authors: A
authors: Schmidt
authors: Department of Medicine, University of California San Diego , La Jolla, California 92093, United States.
authors: Jeramie
authors: J
authors: Watrous
authors: Department of Medicine, University of California San Diego , La Jolla, California 92093, United States.
authors: Mohit
authors: M
authors: Jain
authors: Craig E
authors: CE
authors: Wheelock
authors: Roland
authors: R
authors: Nilsson
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism*; D002247:Carbon Isotopes; D002453:Cell Cycle; D002853:Chromatography, Liquid; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D055432:Metabolomics; D009587:Nitrogen Isotopes
chemical_list: D002247:Carbon Isotopes; D009587:Nitrogen Isotopes