biostudies-literatureChen WWNIAID NIH HHSNCI NIH HHSNIGMS NIH HHS2215-2231https://www.ebi.ac.uk/biostudies/studies/S-EPMC5851482biostudies-literatureUnknown12(10)Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in ∼12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have beNature protocolsRapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites.PMC5851482R01 CA129105T32 GM007753R01 CA103866R37 AI047389Sabatini DMFreinkman EChen WWfalseRapid immunopurification of mitochondria for metabolite profiling and absolute quantification of matrix metabolites.Mitochondria carry out numerous metabolic reactions that are critical to cellular homeostasis. Here we present a protocol for interrogating mitochondrial metabolites and measuring their matrix concentrations. Our workflow uses high-affinity magnetic immunocapture to rapidly purify HA-tagged mitochondria from homogenized mammalian cells in ∼12 min. These mitochondria are extracted with methanol and water. Liquid chromatography and mass spectrometry (LC/MS) is used to determine the identities and mole quantities of mitochondrial metabolites using authentic metabolite standards and isotopically labeled internal standards, whereas the corresponding mitochondrial matrix volume is determined via immunoblotting, confocal microscopy of intact cells, and volumetric analysis. Once all values have be2017-01-01T00:00:00Z2017 Oct2025-04-04T09:53:32.752Z2019-03-26T23:58:06ZS-EPMC58514822953280110.1038/nprot.2017.104