Cardiac Atrial Compartmentalisation Proteomics: A Modified Density Gradient Method to Analyse Endo-lysosomal Proteins

Evidence that lysosomes are important in cardiac physiology and pathology is now emerging. We describe a label-free method suitable for small cardiac tissue biopsies based on a density-separated fractionation protocol, which allows us to study endo-lysosomal (EL) proteins in guinea pig atria. The three most relevant density gradient fractions obtained were analysed using Western Blot, enzyme activity assay and MS/MS peptide analysis (adapted discontinuous Percoll, and sucrose differential density gradient). These fractions comprise whole tissue lysate; sarcoplasmic reticulum (SR), mitochondrial proteins (Mito) (1.3 g/mL); and ‘pure’ EL with negligible contamination from SR or Mito (1.04 g/mL). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther and Gene Ontology pathway analysis showed good coverage of RAB proteins, lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. The most significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and dataset of guinea-pig atrial EL focussed organelle proteomics