期刊
ISCIENCE
卷 24, 期 9, 页码 -出版社
CELL PRESS
DOI: 10.1016/j.isci.2021.102949
关键词
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资金
- Sir Henry Dale Wellcome Trust
- Royal Society Fellowship [109371/Z/15/Z]
- Returning Carers' Fund, Medical Sciences Division, University of Oxford
- Royal Society [109371/Z/15/Z]
- British Heart Foundation [PG/18/4/33521]
- Wellcome Trust Investigator in Science
- Mizutani Foundation
- theWellcome Trust
- Wellcome Trust [109371/Z/15/Z] Funding Source: Wellcome Trust
A label-free proteomics method suitable for small cardiac tissue biopsies was described in this study, allowing for the study of endolysosomal proteins. The analysis of density-separated fractions revealed good coverage of RAB proteins and lysosomal cathepsins in the purified endolysosomal fraction, including cardiac-specific cathepsin D.
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.
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