Dual-Lipa: A sequential multi-omic subcellular landscape map of mouse heart

Spatial multi-omics can provide a unique understanding in molecular organisation and heterogeneity of organs including the heart. Heart function is dependent on the abundance and the spatial arrangement of proteins and lipids. Yet, an integrated multi-omics landscape of the heart at subcellular scale remains unknown. Here, we used a sequential lipid-proteomic analytical pipeline applied to the mouse heart to identify, map, and integrate lipid and protein features, revealing coordinated distinct molecular networks within defined subcellular niches. Here, we benchmark this dual extraction workflow for integrated global proteome-lipidome analyses to demonstrate its performance. We developed conserved subcellular proteome of mouse heart across 14 niches and applied the knowledge of subcellular proteome to spatially resolve the heart lipidome, identifying unique lipid features from different subcellular niches, including mitochondria (e.g., cardiolipin, LPC) and plasma membrane-enriched lipids (e.g., plasmalogens). We identified sex-dependent molecular differences in heart subcellular proteome between male and female, including RNA-protein complexes, mitochondrial calcium handling and immune regulatory pathways. We demonstrate that lipid-protein integrated multi-omics analysis of the heart by dual extraction workflow and mass spectrometry could enable previously unidentified discoveries in heart molecular composition and organisation and spatial cardiac biology. HighlightsO_LIResolved tissue-wide subcellular architecture of cardiac tissue through simultaneous high-resolution proteomics and lipidomics profiling. C_LIO_LISystematically evaluated MS-compatible dual extraction strategies for integrated proteome-lipidome recovery and performance. C_LIO_LIProteomics revealed major sex-dependent differences in mouse left ventricles, primarily involving cellular metabolism, immune regulation, detoxification pathways, and signalling responses. C_LIO_LILipidomics identified strong sex-specific lipid signatures, including differences in omega-3 vs omega-6 fatty-acid containing lipid species, triacylglycerides, and acylcarnitines. C_LIO_LIDeveloped supervised machine-learning models to classify 14 subcellular niches from tissue-wide fractionation proteomics and identified a conserved core subcellular proteome. C_LIO_LIApplied the tissue-wide subcellular proteome model to infer lipid subcellular localisation, revealing mitochondria-enriched lipids (e.g., cardiolipin, LPC) and plasma membrane-enriched lipids (e.g., plasmalogens) in the mouse left ventricle. C_LIO_LIMapped sex-specific subcellular distribution in the heart, highlighting pathways linked to RNA-protein complexes, mitochondrial calcium handling, and immune regulatory networks. C_LI