A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue
Summary: 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...
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| Format: | Article |
| Language: | English |
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Elsevier
2021-09-01
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| Series: | iScience |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2589004221009172 |
| _version_ | 1819129171604930560 |
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| author | Thamali Ayagama Samuel J. Bose Rebecca A. Capel David A. Priestman Georgina Berridge Roman Fischer Antony Galione Frances M. Platt Holger Kramer Rebecca A.B. Burton |
| author_facet | Thamali Ayagama Samuel J. Bose Rebecca A. Capel David A. Priestman Georgina Berridge Roman Fischer Antony Galione Frances M. Platt Holger Kramer Rebecca A.B. Burton |
| author_sort | Thamali Ayagama |
| collection | DOAJ |
| description | Summary: 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. |
| first_indexed | 2024-12-22T08:39:29Z |
| format | Article |
| id | doaj.art-89182a514c6548768b6697ff55f84605 |
| institution | Directory Open Access Journal |
| issn | 2589-0042 |
| language | English |
| last_indexed | 2024-12-22T08:39:29Z |
| publishDate | 2021-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | iScience |
| spelling | doaj.art-89182a514c6548768b6697ff55f846052022-12-21T18:32:16ZengElsevieriScience2589-00422021-09-01249102949A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissueThamali Ayagama0Samuel J. Bose1Rebecca A. Capel2David A. Priestman3Georgina Berridge4Roman Fischer5Antony Galione6Frances M. Platt7Holger Kramer8Rebecca A.B. Burton9University of Oxford, Department of Pharmacology, Oxford, OX1 3QT UKUniversity of Oxford, Department of Pharmacology, Oxford, OX1 3QT UKUniversity of Oxford, Department of Pharmacology, Oxford, OX1 3QT UKUniversity of Oxford, Department of Pharmacology, Oxford, OX1 3QT UKTarget Discovery Institute, University of Oxford, Oxford, OX3 7FZ UKTarget Discovery Institute, University of Oxford, Oxford, OX3 7FZ UKUniversity of Oxford, Department of Pharmacology, Oxford, OX1 3QT UKUniversity of Oxford, Department of Pharmacology, Oxford, OX1 3QT UKBiological Mass Spectrometry and Proteomics Facility, MRC London Institute of Medical Sciences, Imperial College London, London, W12 0NN UKUniversity of Oxford, Department of Pharmacology, Oxford, OX1 3QT UK; Corresponding authorSummary: 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.http://www.sciencedirect.com/science/article/pii/S2589004221009172Porcine cardiologyBiological sciencesBiochemistrySystems biologyOmicsProteomics |
| spellingShingle | Thamali Ayagama Samuel J. Bose Rebecca A. Capel David A. Priestman Georgina Berridge Roman Fischer Antony Galione Frances M. Platt Holger Kramer Rebecca A.B. Burton A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue iScience Porcine cardiology Biological sciences Biochemistry Systems biology Omics Proteomics |
| title | A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue |
| title_full | A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue |
| title_fullStr | A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue |
| title_full_unstemmed | A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue |
| title_short | A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue |
| title_sort | modified density gradient proteomic based method to analyze endolysosomal proteins in cardiac tissue |
| topic | Porcine cardiology Biological sciences Biochemistry Systems biology Omics Proteomics |
| url | http://www.sciencedirect.com/science/article/pii/S2589004221009172 |
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