To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.
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The authors thank Neil Howell and Christen Anderson for helpful comments, and Paul Haynes and Ross Hoffman for technical advice.
MitoKor is a for-profit biotechnology company that has focused on using the mitochondrion as a platform for drug discrovery. Some proteins disclosed in the article are potential molecular drug targets for which patent applications have been, or will be, filed.
Metrizamide gradient purified human heart mitochondria are highly depleted of plasma membrane, cytosol and organellar markers. (PDF 997 kb)
The purity of metrizamide purified mitochondria (lanes 3) compared to the initial human heart homogenate (lanes 1) and differential centrifugation purified mitochondria (lanes 2) was assessed. Equivalent loads of 25 μg (Panels A. B. and C.) or 90 μg (Panel D.) of each sample were separated by 4-12% NuPAGE. Western analysis was carried out with antibodies reactive toward proteins from various cellular locations.
Panel A shows Coomassie staining of protein in each fraction. Panel B. depicts the enrichment of components of the electron transport chain. Complex V was detected with Molecular Probes antibody cat.# A-21350 to ATPase alpha. Complex III was detected with Molecular Probes antibody cat.# A-21362 to Core I. Complex I was detected with Molecular Probes antibody cat.# A -21344 to NDUFA9. Complex IV was detected with Molecular Probes antibody cat.# A-21348 specific for Cox4. Further analysis of mitochondria specific markers is shown in Panel C. HSP 60, a matrix protein, was visualized with an antibody from Stressgen cat.# SPA-806. VDAC, a marker of the outer mitochondrial membrane, was detected with CalBioChem antibody cat.# 529538. Rab-11, after LC/MS/MS identification described in this paper, was confirmed to localize specifically to mitochondria using BD Transduction Labs antibody cat.#610656. These Western analyses demonstrate a good enrichment and integrity of mitochondria following metrizamide purification.
Reduced reactivity was detected in the DC mitochondria (lanes 2) and metrizamide mitochondria (lanes 3) by varous protein markers for nonmitochondrial proteomes, shown in Panel D. Dynamin II (cytosol/plasma membane), detected with BD Transduction Labs antibody cat.#D27120, Grp 95 and Grp 76 which contain the KDEL epitope (endoplasmic reticulum), detected with Stressgen antibody cat.#SPA-827, LAMP-1 (lysosome), detected with Santa Cruz Biotechnology antibody cat.#sc -17768, and alpha actin (cytosol), detected with Sigma antibody cat.#A2172 are depicted. Filters shown in Panel D were scanned on a Fluor-S Max Imaging System (BioRad) and quantitated using QuantityOne software. By this analysis, dynamin II was depleted by 95%, LAMP-1 by 98%, a-actin by 99%, Grp95 by 97%, and Grp76 by 95% in the metrizamide purified mitochondria relative to the initial human heart homogenate.
Plots of gel slice vs average molecular weight value of SEQUEST hits for sucrose density gradient fractions and pellet. (PDF 23 kb)
Distribution of the five complexes of the oxidative phosphorylation machinery within the sucrose gradient plotted as the number of SEQUEST matches (according to the threshold criteria in the Supplementary Information) to tryptic peptides of each of their subunits. Compare with the plot of Western intensities in Taylor et al. J. Proteome Research 1, 451–458 (2002). The distribution of selected proteins discussed in the text throughout the gradient is shown. Abbreviations: PHB's, prohibitins; SSBP, single stranded mitochondrial DNA binding protein; mtRBP's, mitochondrial ribosomal DNA binding proteins; cMDH, cytosolic malate dehydrogenase; GK, glycerol kinase; ICDH, isocitrate dehydrogenase; MXN, metaxin 2; mtMDH, mitochondrial MDH; CS, citrate synthase; AC, aconitase; FH, fumarate hydratase; LDH, lactate dehydrogenase; ADH, aldehyde dehydrogenase. (PDF 13 kb)
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Taylor, S., Fahy, E., Zhang, B. et al. Characterization of the human heart mitochondrial proteome. Nat Biotechnol 21, 281–286 (2003). https://doi.org/10.1038/nbt793
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