A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue
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 endolyso...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Journal article |
| Language: | English |
| Published: |
Elsevier
2021
|
| _version_ | 1797096931579658240 |
|---|---|
| author | Ayagama, T Bose, SJ Capel, RA Priestman, DA Berridge, G Fischer, R Galione, A Platt, FM Kramer, H Burton, RAB |
| author_facet | Ayagama, T Bose, SJ Capel, RA Priestman, DA Berridge, G Fischer, R Galione, A Platt, FM Kramer, H Burton, RAB |
| author_sort | Ayagama, T |
| collection | OXFORD |
| description | 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 | 2024-03-07T04:48:30Z |
| format | Journal article |
| id | oxford-uuid:d4269bc2-5584-460a-922d-221945180456 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T04:48:30Z |
| publishDate | 2021 |
| publisher | Elsevier |
| record_format | dspace |
| spelling | oxford-uuid:d4269bc2-5584-460a-922d-2219451804562022-03-27T08:16:20ZA modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissueJournal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:d4269bc2-5584-460a-922d-221945180456EnglishSymplectic ElementsElsevier2021Ayagama, TBose, SJCapel, RAPriestman, DABerridge, GFischer, RGalione, APlatt, FMKramer, HBurton, RABThe 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. |
| spellingShingle | Ayagama, T Bose, SJ Capel, RA Priestman, DA Berridge, G Fischer, R Galione, A Platt, FM Kramer, H Burton, RAB A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue |
| 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 |
| work_keys_str_mv | AT ayagamat amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT bosesj amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT capelra amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT priestmanda amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT berridgeg amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT fischerr amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT galionea amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT plattfm amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT kramerh amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT burtonrab amodifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT ayagamat modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT bosesj modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT capelra modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT priestmanda modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT berridgeg modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT fischerr modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT galionea modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT plattfm modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT kramerh modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue AT burtonrab modifieddensitygradientproteomicbasedmethodtoanalyzeendolysosomalproteinsincardiactissue |