Cellular lipid metabolism is a complex network process comprising dozens of enzymes, multiple organelles and more than a thousand lipid species. Tracing metabolic reactions in this network is a major technological and scientific challenge. Using a click-chemistry mass spectrometry reporter strategy, we have developed a specific, highly sensitive and robust tracing procedure for alkyne-labeled lipids. The method enables sample multiplexing, which improves sample comparison. We demonstrate this by a time-resolved analysis of hepatocyte glycerolipid metabolism with parallel quantitative monitoring of 120 labeled lipid species. The subfemtomole sensitivity enabled a single cell analysis of fatty acid incorporation into neutral and membrane lipids. The results demonstrate the robustness of lipid homeostasis at the single cell level.
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