CerS2 dependent Sphingolipid - protein interactions in rat beta cell line Ins1E

To define the beta cell Sphingolipid-protein interactome, we performed a chemoproteomic screen in which a chemically modified SL precursor (photoactivatable and clickable sphingosine, pacSph) was fed to cells and rapidly incorporated into de novo synthesized SLs. Ultraviolet (UV) irradiation covalently crosslinked cellular SL-protein complexes, allowing for cell lysis under harsh conditions, pulldown and subsequent MS-based identification and quantification of SL interacting proteins. CrispR/Cas9 knockout of the SL specific catabolic enzyme Sgpl1 ensured that only SLs are labelled with pacSph. Furthermore, stable isotope labelling allowed direct comparison of SBPs in several cell lines in the same MS run.