A. Percoll gradient improved sample purity. Equal amounts (50μg) of mitochondria either with or without further enrichment by percoll purifications were separated by SDS-PAGE followed by immunoblotting with subcellular marker proteins including, LAMP-1 (lysosome), DHPRα1 (sacrolemma [SM]), GRP78 (endoplasmic reticulum [ER]), NuMA (nucleus), as well as LDH activities (left middle panel). Note that percoll effectively eliminated the contaminations from lysosome, sarcrolemmal membrane, cytosol, and nucleus. Percoll step had no detectable effect on mitochondrial proteins (right panel), including HSP60 and GRP 75 (matrix), VDAC (outer membrane) and ANT (inner membrane). Although percoll removed some ER proteins, the ER marker GRP78 remained visible in the post-percoll gradient fraction (left bottom panel). Ponceau S-stained nitrocellulose membranes document equal loading (right bottom panel). B. Percoll-gradient removed mitochondria with broken outer membranes. The mitochondrial integrity was assessed by cytochrome c oxidase activity in the presence and absence of 0.1% DDM, which broke outer membranes and established maximal (100%) cytochrome c oxidase activity. The percoll gradient effectively reduced broken mitochondria from 15% to 4% of the total, rendering a preparation with improved integrity. C. Functional validation of purified mitochondria. Mitochondrial function was documented by respiratory ratio (>5) and intact mitochondrial membrane potential. Purified mitochondria (0.4 mg/ml) were added to KCl buffer (140 mM KCl and 10 mM HEPES, pH 7.4 with Tris). At the arrows, 5 mM P_i and complex I substrates [pyruvate (Pyr), malate (Mal), and glutamate (Glu), each 1.5 mM] were added, followed by addition of ADP at the indicated concentrations. The top tracing shows that membrane potential dissipated transiently after each ADP addition but fully recovered after a time delay proportional to the amount of added ADP. The bottom tracing shows that O₂ consumption (i.e., buffer PO₂ decrease) also accelerated transiently during ADP phosphorylation. At the end, alamethicin, a non-specific membrane permeabilizing agent (Ala; 5 μg/ml), was added to induce complete dissipation and maximum swelling for calibration purposes. D. Assessment of mitochondrial functionality by swelling assay. Calcium challenge leads to mitochondrial swelling, which could be prevented by pretreatment with cyclosphorin A (CsA). E. The morphology of the isolated mitochondria by electron microscope. Arrows in left panel indicate that ruptured mitochondria and non-mitochondrial membranes were virtually absent following percoll (magnification 19,000x).

A. Genomic distribution of genes encoding proteins identified in murine cardiac mitochondria. Frequency percentage was calculated by the number of identified proteins localized in individual chromosomes divided by the number of total proteins identified in this study. B. Distribution of database sources on identified murine cardiac mitochondrial proteins and the murine proteome. The distribution of identified IPI entries across the underlying databases was based on an entry having at least one reference in the source databases in the order UniProtKB/Swiss-Prot, UniProtKB/TrEMBL, Vega, Havana, ENSEMBL, RefSeq. The murine proteome is represented by all entries in IPI murine.

A. Transmembrane domain and transit peptides were predicted using TMHMM 2.0 and TargetP1.1. Among 940 identified proteins, 195 proteins (20.7%) had one or more transmembrane domains; in addition, 469 out of 940 proteins had mitochondrial transit peptide sequences (inlet). B-D Physiochemical property classifications of identified proteins. B. Molecular weight (MW, in kDa) analyses; C. Isoelectric focusing point (pI) distributions; D. Grand average hydrophobicity (GRAVY) distributions. Molecular weight, isoelectric point, and grand average hydropathicity value were calculated using the online ProtParam tool available through ExPASY.

Individual protein was assigned to their respective function cluster(s) and spatial location(s) according to the Gene Ontology Annotations database (GOA), the InterPro database, OMIM, and author initiated Pubmed search. The y axis represents the subcellular compartment(s) where a protein resides, whereas the x axis indicates the function cluster it belongs to. Our proteomic approach identified a total of 940 unique proteins residing in the cardiac mitochondria. In addition to mitochondria, many proteins reside in one or more other cellular locations. Among 940 proteins, 169 were also found in cytoplasm, 50 in ER, 20 in Golgi, 144 in sacrolemma or other membrane fractions, 16 in lysosome, 24 in perixosome, 57 in nucleus, and 137 in extra-cellular space. Furthermore, 47 out of 940 proteins were assigned to more than one function cluster(s). Arrows point to three examples, including the eukaryotic peptide chain release factor that is experimentally identified in the cardiac mitochondria; this protein was also previously found in the cytoplasm; and this protein belongs to the function cluster of DNA/RNA/Protein synthesis.

Target validation using immunoblottings was conducted using two independent samples (S1 and S2). The upper panels confirm the expression of five proteins in mitochondria documented by previous studies [11,12,15]; the bottom panels validate the mitochondrial localization of five proteins identified in the present study but none of them was previously reported as mitochondria proteins [11,12,15]. Validations of identified proteins were also carried out by confocal immunochemistry, data not shown.