Identification of specific lipid-protein interactions in dividing cells using lipid-trap mass spectrometry

Cells actively maintain complex lipidomes that encompass thousands of lipids, however, many of the roles of these lipids remain unexplored. Specific interactions between lipids and membrane proteins are a likely reason for the evolutionary conservation of complex lipidomes. We report the development of a technique, named lipid-trap mass spectrometry (LTMS), to systematically study protein-lipid interactions directly captured from mammalian cells. LTMS uses immunoprecipitation of GFP-tagged proteins expressed in HeLa, followed by lipidomic analysis of lipids bound to the GFP-tagged protein. We applied LTMS to cell division to illustrate the technique. We chose this process because membranes regulate their lipid composition as they undergo major changes during cytokinesis and many cytokinetic proteins, including RACGAP1 and ESCRTIII components CHMP4B and CHMP2A, are membrane-associated. Using LTMS, we found that RACGAP1 and CHMP4B associate with specific lipid species in dividing compared to non-dividing cells. We expand our understanding of lipid diversity during cell division and present a general approach to explore lipid-protein interactions to further our understanding of the roles of lipids in mammalian cells.