Lipids play essential roles in regulating various biological processes through functional interactions with integral or peripherally associated proteins and protein complexes. While mass spectrometry (MS)-based strategies have enabled global lipidome and proteome identification and quantitative profiling, high-throughput strategies to investigate the lipid-protein ‘interactome’ at the systems level are currently lacking. Here, I will first describe the development of advanced ‘shotgun’ lipidomics data acquisition workflows, including the use of ion-mobility for selective enrichment of low abundance lipid classes, and UV-photodissociation MS/MS to enable complete lipid structural characterisation [e.g., Anal. Chem. 2024, 96, 12296-12307.; Anal. Bioanal. Chem. 2020, 412, 2339–2351.], and their application to characterise a novel cause of autosomal regressive multisystem mitochondrial disease resulting from deleterious variants in cardiolipin synthase 1 (CRLS1) [Human Mol. Genetics. 2022, 31, 3597-3612.]. Next, using an integrated ‘multi-omics’ workflow for comprehensive lipidome and proteome profiling, I will report that the knockout of non-mitochondrial genes in peroxisome, endoplasmic reticulum (ER) and Golgi organelles also results in mitochondrial dysfunction, caused by unexpected alterations in ether-glycerophospholipid metabolic pathways [Nature. Cell Biol. 2024, 26, 57-71]. Finally, to investigate the lipid–protein interaction relationships in these systems, a novel Co-Fractionation Multi-Omics Mass Spectrometry (CF-MOMS) strategy will be described to characterise global alterations in the lipid-protein ‘interactome interactomes in WT and CRLS1KO cell lines, and to highlight the functional involvement of cardiolipin-protein interactions in maintaining the assembly/stability and activity of mitochondrial membrane proteins.