Abstract
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.
Highlights
The concept of understanding proteins from a compartmentalization perspective, their interconnected properties, and dynamic distribution in health and disease is critical for deciphering the phenotype of a cell (Larance and Lamond, 2015)
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 (LCMS/MS) analysis
Summary
The concept of understanding proteins from a compartmentalization perspective, their interconnected properties, and dynamic distribution in health and disease is critical for deciphering the phenotype of a cell (Larance and Lamond, 2015). Significant advances in mass spectrometry-based proteomics allow scientists to achieve multidimensional measurements of proteins with greater efficiency, enabling for example the generation of more detailed maps of the human proteome (Kim et al, 2014). Relative quantification methods of samples include label-free quantification (Hoffert et al, 2006), in vivo metabolic stable isotope labeling (Wang et al, 2002), stable isotope labeling using chemical tags that are covalently attached in vitro, tandem mass tags, and isobaric tags for relative and absolute quantification (recently reviewed by Larance and Lamond (2015))
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