Proteomic Analysis of Purified Dog Cardiac Sacroplasmic Reticulum Membrane Subcompartments

Abstract

Ca2+ homeostasis primarily involves two major morphological subdivisions of the sarcoplasmic reticulum (SR): junctional (jSR) cisterna that appose sarcolemma, and free (fSR) tubules surrounding myofilaments. Biochemical isolation of jSR and fSR membranes involves selective increases in density of SR membrane vesicles using the SR Ca2+ pump (SERCA2a) (Jones and Cala, J. Biol. Chem. 1981). The purpose of the current study was to comprehensively characterize cardiac SR subproteomes. Purified SR membranes were solubilized, trypsinized and analyzed by LC-MS/MS on a LTQ-XL mass spectrometer. Abundant species were fragmented with collision-induced dissociation (CID). Data analysis was performed using Proteome Discoverer 1.1 (Thermo) which incorporated the Mascot algorithm (Matrix Science). In replicate experiments, a total of 199 junctional SR and 191 free SR proteins were identified from 16,344 MSMS spectra with protein false discovery rate (FDR) at 0.8% and peptide FDR at 0.0%. SERCA2a was the most abundant protein in both fractions, as expected, and virtually all of the known cardiac SR proteins were also identified. In jSR, the major 4 components of the Ca2+-release complex were found exclusively, including calsequestrin-2, ryanodine receptor 2 and 3, triadin, and junctin. Another 57 proteins were also identified as specific to this subcompartment. In fSR, 53 proteins were exclusively identified. Relative enrichments in one of the SR subcompartments were found for an additional 86 proteins, and 52 additional proteins were identified in the cardiac SR, without obvious subcompartment origin. SR proteins that showed less specificity were mitochondrial proteins (more enriched in the lower-density jSR), ER chaperones (roughly evenly distributed), lipid-metabolizing enzymes, and filamentous proteins. Proteomic analyses of classical cardiac SR subfractions extends our understanding of the cardiac secretory compartments, and serves as foundation for future exploration and understanding of cardiac cell biology.