文章基本信息

标题：A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue
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作者：Thamali Ayagama ; Samuel J. Bose ; Rebecca A. Capel 等
期刊名称：iScience
印刷版ISSN：2589-0042
出版年度：2021
卷号：24
期号：9
页码：1-21
DOI：10.1016/j.isci.2021.102949
语种：English
出版社：Elsevier
摘要：SummaryThe 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.Graphical abstractDisplay OmittedHighlights•Method to analyze endo-lysosomal proteins from guinea pig cardiac atrial tissue•Endolysosome organelle proteomics using techniques applicable for non-cardiac tissue•We identified endo-lysosomal marker proteins such as Rab7A, VPS29, RILP, and LAMTOR1-5Porcine cardiology; Biological sciences; Biochemistry; Systems biology; Omics; Proteomics