A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue.

Ayagama, Thamali, Bose, Samuel J ORCID: 0000-0003-2922-3075, Capel, Rebecca A, Priestman, David A, Berridge, Georgina, Fischer, Roman ORCID: 0000-0002-9715-5951, Galione, Antony, Platt, Frances M, Kramer, Holger and Burton, Rebecca AB ORCID: 0000-0002-0904-3862 (2021) A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue. iScience, 24 (9). 102949-.

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Official URL: http://dx.doi.org/10.1016/j.isci.2021.102949

Abstract

The importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Item Type:	Article
Uncontrolled Keywords:	Biochemistry, Biological sciences, Omics, Porcine cardiology, Proteomics, Systems biology
Divisions:	Faculty of Health and Life Sciences Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	09 Apr 2024 14:19
Last Modified:	09 Apr 2024 15:41
DOI:	10.1016/j.isci.2021.102949
Related URLs:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3180243