title: Bifunctional Sphingosine for Cell-Based Analysis of Protein-Sphingolipid Interactions.
abstract: Sphingolipids are essential structural components of cellular membranes and are crucial regulators of cellular processes. While current high-throughput approaches allow for the systematic mapping of interactions of soluble proteins with their lipid-binding partners, photo-cross-linking is the only technique that enables for the proteome-wide mapping of integral membrane proteins with their direct lipid environment. Here, we report the synthesis of a photoactivatable and clickable analog of sphingosine (pacSph). When administered to sphingosine-1-phosphate lyase deficient cells, pacSph allows its metabolic fate and the subcellular flux of de novo synthesized sphingolipids to be followed in a time-resolved manner. The chemoproteomic profiling yielded over 180 novel sphingolipid-binding proteins, of which we validated a number, demonstrating the unique value of this technique as a discovery tool. This work provides an important resource for the understanding of the global cellular interplay between sphingolipids and their interacting proteins.
authors.affiliation: European Molecular Biology Laboratory , Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Per
authors.initials: P
authors.lastname: Haberkant
authors.affiliation: European Molecular Biology Laboratory , Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Stein
authors.affiliation: European Molecular Biology Laboratory , Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Doris
authors.initials: D
authors.lastname: Höglinger
authors.affiliation: Heidelberg University Biochemistry Center , Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
authors.forename: Mathias J
authors.initials: MJ
authors.lastname: Gerl
authors.affiliation: Heidelberg University Biochemistry Center , Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Brügger
authors.affiliation: Laboratory for Lipid Biochemistry and Protein Interactions, Department of Cellular and Molecular Medicine, KU Leuven , B-3000 Leuven, Belgium.
authors.forename: Paul P
authors.initials: PP
authors.lastname: Van Veldhoven
authors.affiliation: European Molecular Biology Laboratory , Genome Biology Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Jeroen
authors.initials: J
authors.lastname: Krijgsveld
authors.affiliation: European Molecular Biology Laboratory , Structural and Computational Biology Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Anne-Claude
authors.initials: AC
authors.lastname: Gavin
authors.affiliation: European Molecular Biology Laboratory , Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Schultz
mesh_terms: D006801:Humans; D015394:Molecular Structure; D040081:Protein Array Analysis; D011485:Protein Binding; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results; D013107:Sphingolipids / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000737:chemistry*
chemical_list: D013107:Sphingolipids; D013110:Sphingosine