A photoactivatable chemical lipidomics approach for local sphingolipid metabolic analysis

In eukaryotic cells, lipid metabolism is tightly regulated depending on the subcellular localization, which is essential for maintaining lipid homeostasis. However, understanding compartmentalized lipid metabolism remains challenging due to limited availability of suitable techniques. In this study, we present a chemical lipidomics approach that combines photoactivatable probes with high resolution mass spectrometry and stable-isotope labelling to analyze lipid dynamics at subcellular resolution. We applied this method to analyze the metabolism of 1-deoxysphingolipid (DoxSL), a non-canonical lipid species linked to various metabolic diseases and neuropathy, whose metabolism remains largely unexplored. Using the photoactivatable probes, we selectively delivered 1-deoxysphinganine, a key DoxSL intermediate, to mitochondria upon photo-illumination and subsequently analyzed its local metabolic products over time. Our data show that most 1-deoxysphinganine delivered to mitochondria is rapidly converted into 1-deoxyceramides, while only a small fraction forms oxidized products. Further lipidomic analysis revealed that 1-deoxyceramides are transported to the extracellular space and that DoxSL is also present in mouse and human serum samples. In summary, we developed novel probes to track lipid dynamics with high spatiotemporal resolution in a non-invasive manner and provided new insights into sphingolipid metabolism.