Oral Presentation AUS-oMicS 2025

Development of a Co-Fractionation Mass Spectrometry Based Strategy for Lipid-Protein Interactome Analysis (#82)

Liuyu Peng 1 , Richard G Lee 2 3 4 , Nichollas E Scott 5 , Aleksandra Filipovska 1 2 3 4 , Gavin E Reid 1 6 7
  1. School of Chemistry, The University of Melbourne, Parkville, VIC, Australia
  2. The Kids Research Institute Australia, Nedlands, WA, Australia
  3. Harry Perkins Institute of Medical Research, Nedlands, WA, Australia
  4. ARC Centre of Excellence in Synthetic Biology, Centre for Medical Research, QEII Medical Centre, The University of Western Australia, Nedlands, WA, Australia
  5. Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, Parkville, VIC, Australia
  6. Department of Biochemistry and Pharmacology, The University of Melbourne, Parkville, VIC, Australia
  7. Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC, Australia

Lipids, as fundamental components of cellular membranes, play crucial roles in maintaining plasma membrane and organellar structures, as well as regulating various biological processes through functional interactions with integral or peripherally associated proteins. Although the importance of many specific protein-lipid interactions has been elucidated, methods for the identification and functional characterization of the lipid-protein ‘interactome’ on a global scale are currently lacking. Here, we have developed a co-fractionation mass spectrometry (CF-MS) technique for lipid-protein ‘interactome’ analysis, employing size exclusion chromatography (SEC) coupled with mass spectrometry (MS), and have applied this strategy to characterise alterations in membrane lipid-protein interactomes in a cardiolipin synthase-associated multi-system mitochondrial disease system. Non-denaturing membrane protein-lipid complex solubilization was carried out under an optimised mild detergent membrane solubilisation environment prior to SEC co-fractionation. To confirm the efficiency of solubilization and stability of solubilized membrane protein complexes and lipid-protein complexes, blue native gel, and quantitative MS was utilized. Co-fractionated lipid-protein interactions were then identified using an integrated lipid and protein extraction method followed by LC-MS/MS based lipidome and data-independent acquisition (DIA) proteome analysis. CF-MS lipid-protein interactome analysis were conducted on wild-type (WT) and cardiolipin synthase knockout (CRLS1-KO) CAL51 cell lines. Results showed that cardiolipin species are co-fractioned with intact mitochondria respiratory chain protein complexes and supercomplexes in WT cell lines. Conversely, significant disassembly of complex III subunits and mild disassembly of complex I subunit from mitochondria supercomplexes and were observed in the CRLS1-KO cell line, highlighting the functional involvement of lipid-protein interactions in maintaining the assembly/stability and activity of mitochondrial membrane proteins.