www.nature.com/naturebiotechnology • MARCH 2003 • VOLUME 21 • nature biotechnology

Characterization of the human heartmitochondrial proteome

Steven W. Taylor1*, Eoin Fahy1, Bing Zhang1, Gary M. Glenn1, Dale E. Warnock1, Sandra Wiley1, Anne N. Murphy1,Sara P. Gaucher2, Roderick A. Capaldi3, Bradford W. Gibson2, and Soumitra S. Ghosh1

Published online 18 February 2003; doi:10.1038/nbt793

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an exten-sive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human hearttissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individualprotein components were separated by one-dimensional PAGE. Total in-gel processing and subsequentdetection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct proteinidentifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. Thecoverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrialmembrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, andprotein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteinshave not been defined. This database of proteins provides a comprehensive resource for the discovery ofnovel mitochondrial functions and pathways.

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Mitochondria are ideal targets for global proteome analysis becausethey have a manageable level of complexity as a consequence of theirapparent prokaryotic ancestry. In addition, they are centrally involvedin a large number of human disorders. Aside from their bioenergeticfunction, mitochondria regulate cell death, modulate ionic homeosta-sis, oxidize carbohydrates and fatty acids, and participate in numerousother catabolic and anabolic pathways. Consequently, mitochondrialdysfunction can have grave consequences that range from defects inenergy metabolism to etiologically complex diseases with a mitochon-drial association, including Alzheimer’s and Parkinson’s diseases, can-cer, type 2 diabetes, cardiovascular disease1, and osteoarthritis2. Themitochondrial connection to these major degenerative diseases hasdriven experimental efforts to define the mitochondrial proteome andto discover new molecular targets for drug development and thera-peutic intervention.

MiTOchondria Project (MITOP), a publicly accessible database,lists 340 mitochondrial genes and proteins for humans3, well short ofthe approximately 1,500 that have been predicted4. In the currentinvestigation, we have used mass spectrometric–based protein iden-tification to further characterize the mitochondrial proteome. Wechose human heart tissue as the source of mitochondria because (i) the organelle can be isolated at high purity (see SupplementaryFig. 1 online) and in high yield5,6, (ii) there are extensive databases ofhuman proteins available for bioinformatic analysis, (iii) a substan-tial amount of tissue can be collected compared with other speciesfor which genomes are known (for example, mouse), and (iv) theproteins identified will be most relevant for the study of human dis-ease. Here we report the most comprehensive database of mitochon-drial and mitochondria-associated proteins to date, with a total of615 distinct protein identifications (see Supplementary Tables 1 and2 online). To our knowledge, the largest proteomic database previ-

ously generated in a single study for a highly purified organelle is the311 proteins of the human spliceosome7 from nuclear extracts.

Results and DiscussionWe recently described an approach to elucidate the mitochondrial pro-teome that uses sucrose density gradient fractionation to separateintact protein complexes5 followed by one-dimensional polyacry-lamide gel electrophoresis (PAGE) and high-throughput peptide massfinger printing (PMF)6. This strategy has been refined in the currentinvestigation by performing total in-gel processing on 11 sucrose frac-tions and the n-dodecyl-β-D-maltoside–insoluble pellet (SDS-PAGEimages are available available on the authors’ website; see URL inExperimental protocol) and by using liquid chromatography–tandemmass spectrometry (LC-MS/MS) as an additional protein identificationtool (see Supplementary experimental protocol online). Strategies usedto integrate the large quantity of mass spectral data (data available onthe authors’ website; see URL) and to validate protein identifications,including the use of molecular weight–gel migration correlations (seeSupplementary Fig. 2 online) and synthetic peptides (more informa-tion is available on the authors’ website; see URL)), are also described inthe Supplementary experimental protocol online, as are the bioinfor-matic approaches including database considerations for protein identi-fication algorithms, the differentiation of protein isoforms (seeSupplementary Table 3 online), and motif analyses.

General properties of the identified proteins. The distribution ofpI values over the range 4–11 is shown in Figure 1A. Recently, similarhistograms were computed for the proteomes of several prokaryoteswith fully sequenced genomes8. It was found that integral membraneproteins clustered around pI 8.5–9.0, whereas cytosolic proteins clus-tered around pI values of 5–6. In eukaryotes, there was also a thirdcluster with pI values around 7.0. Our experimental data indicates

1MitoKor, 11494 Sorrento Valley Road, San Diego, California 92121. 2Buck Institute for Age Research, Novato, California 94945. 3Institute for Molecular Biology,University of Oregon, Eugene, Oregon 97403. *Corresponding author (taylors@mitokor.com).

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that the distribution of the predicted pI values of mitochondrial pro-teins is typical of a eukaryotic cell, with a roughly trimodal distribu-tion. An overrepresentation of basic proteins in the mitochondrion,compared with the pI histograms for the eukaryotic proteins, mayrepresent both the cationic characteristics necessary for efficientimport into their mitochondrial locations and the high proportion ofintegral membrane proteins identified here. The latter have a higherproportion of basic residues to promote stability through favorableelectrostatic interactions8.

Proteins in the 20–30 kDa range were the most prevalent in ourdatabase as shown in Figure 1B, which illustrates the molecular weightdistribution of all identified proteins. Comparatively few had molecu-

lar weights greater than 150 kDa. This observation may mean thatlower-molecular-weight proteins dominate the mitochondrial pro-teome, or it could be an artifact resulting from lower recovery of pep-tides from higher molecular weight proteins in polyacrylamide gels.However, of the large proteins identified, cytoskeletal myosin-β heavypolypeptide (223 kDa) and spectrin-αII (285 kDa) were representedby 40 peptides and 5 peptides, respectively, in the gel lane containingthe solubilized pellet (see below). These data indicate that there werefew impediments to the identification of proteins in the higher molec-ular weight range, and that mitochondria contain a small proportionof high-molecular-weight proteins.

The hydrophobicities of all identified proteins were estimatedwith the Kyte-Doolittle algorithm9 (Fig. 1C). For comparison, wehave also plotted subsets of proteins belonging to theRNA/DNA/protein synthesis family (which tend to be morehydrophilic) and proteins involved in transport across membranes(which tend to be more hydrophobic) on the same graph. Because ofthe size of the data set, we have not undertaken a regional analysis ofhydrophobic and hydrophilic regions in individual proteins. Thesymmetrical distribution of values (Fig. 1C) indicates that proteinswith highly variable degrees of hydrophobicity are represented, thusgiving us confidence that the recovery of membrane-bound proteinswas not biased as it is when two-dimensional PAGE is used for elec-trophoretic resolution6.

Functional classification. On the basis of GenBank annotationand hidden Markov model (HMM) motif-based searches10, the dataset of the identified proteins can be subdivided into two sets: thosethat can be assigned into known functional classes (81%) and thoseto which no functionality has been ascribed (19%). The distributionof the functionally classified proteins is shown in Figure 2 and is list-ed by subclass in Supplementary Table 1 online.

The oxidative phosphorylation (OXPHOS) machinery. Subunitsof the five complexes that constitute the oxidative phosphorylationmachinery represent a major group of functionally classified mito-chondrial proteins. We have obtained extensive coverage (91%) ofthese proteins, including 42/45 complex I subunits, 4/4 complex IIsubunits, 8/11 complex III subunits, 12/13 complex IV subunits, and15/16 complex V subunits, and we have identified several subunitisoforms (see Supplementary Table 3 online). Notably, we identifiedhuman homologs of three newly discovered complex 1 subunits inbovine heart mitochondria: GRIM-19, a regulator of cell death11, andESSS and B14.7 (ref. 12), which were defined as ‘neuronal protein’(GenBank accession number GI 13938442) and ‘unknown’ (GI17455445), respectively12.

Signaling proteins. This functional class includes guanine-, GTP-,and RAS-related proteins, kinases/phosphatases, protease inhibitors,receptors, and other proteins that have a role in intra- and intercellu-lar communication. We identified 25 guanine/GTP/RAS-relatedproteins and 9 kinases/phosphatases, indicating that extensive sig-naling pathways for intraorganellar communication and regulationoperate in mitochondria, although the majority of the pathways havenot been elucidated. Most of the GTP-binding proteins were relatedto RAS, and they are likely to be involved in signaling or membranefusion processes. Pyruvate dehydrogenase kinase was detected by asingle peptide in the pellet. We also identified seven isoforms of theannexin family of proteins, which show a calcium-dependent

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Figure 1. Classification of proteins based on physiochemical properties.(A) pI distribution of identifications, over increments of 0.4 pH units.(B) Molecular weight distribution of protein identifications, using 10 kDaincrements. (C) Hydrophobicity distribution of identified proteins, usingmean Kyte-Doolittle amino acid values. As well as the distribution for theentire set of 615 proteins, the RNA/DNA/protein synthesis and transportersand channels classes are also plotted as examples of hydrophilic andhydrophobic sub-groups, respectively.

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interaction with phospholipid membranes, as well as a tyrosine 3-mono-oxygenase/tryptophan 5-mono-oxygenase activation pro-tein that belongs to the 14-3-3 family. Members of the 14-3-3 familyare multifunctional proteins that play roles in various cell-signalingevents and BAD-mediated apoptosis13.

Transporters and channels. Twelve distinct identifications of car-rier proteins were found that belong to this functional group. Asgauged by the relative confidence of their identifications, the mostabundant proteins included phosphate carrier isoform B, oxoglu-tarate/malate carrier, carnitine-acylcarnitine translocase and ade-nine nucleotide translocase isoform 1 (ANT1). ANT1 is a highlyhydrophobic inner-membrane carrier and is the principal proteinin mitochondrial preparations6, but it is not observed on two-dimensional gels14–17. Aralar and citrin, recently determined to beCa2+-stimulated aspartate/glutamate carriers, were also found18.Another 31 proteins were classified as channels/transporters,including the three voltage-dependent anion channel (VDAC) iso-forms of the outer mitochondrial membrane. We also identified thesarcoplasmic reticulum associated calcium ATPase 2 (Serca2),which is known to be localized in areas of contact between the sar-coplasmic reticulum and the outer mitochondrial membrane19. Na+,K+-ATPase subunit α (two isoforms) was detected; it may representa site of contact of the plasma membrane with the mitochondrionin a manner analogous to Serca2.

RNA, DNA, and protein synthesis. Proteins involved in RNA,DNA, and protein synthesis represented the next major category. Thesingle-stranded mitochondrial DNA-binding protein was found inthe dense sucrose gradient fractions, suggesting that its associationwith mitochondrial DNA persisted within the gradient. Five proteinsinvolved in transcription were identified, of which the mitochondrialtranscription factor-1 showed the best tryptic peptide coverage. Ofthe large (39 S) and small (29 S) subunits of the human mitochondr-ial ribosome, 15 and 20 protein subunits were identified, respectively.The mitochondrial ribosomal proteins were found almost exclusivelyin sucrose gradient fractions 1 and 2, indicating that the ribonucleo-protein complex sedimented essentially intact through the sucrosegradient. Recent investigations of the bovine mitochondrial ribosomesuggest that the large subunit contains approximately 48 proteins20,21

and the smaller subunit comprises 29 proteins22,23, indicating that ourcoverage is approximately 30% and 70%, respectively. Of the newlyidentified mitochondrial ribosomal proteins isolated from bovine tis-sue that were reported in those studies, we verified human homologsfor L1, L9, L13, L18, L20, L27, L49, L53, L56, S2, S6, S11, S16, S18a,S18b, S29, S33, S35, and S36. The protein DAP3 (death-associatedprotein, also known as MRPS29) is significant because it is associatedwith apoptosis23. In addition to the mitochondrial ribosomal pro-teins, three elongation factors were identified, with the major proteinbeing Tu translation elongation factor or TUFM.

Metabolism. The tricarboxylic acid (TCA) cycle (plus pyruvatedehydrogenase) was represented by 25 distinct protein subunits orisoforms of enzymes in the pathway. Based on recent reports of asupramolecular complex involving various dehydrogenases inyeast24, we examined the sucrose fractions in which the TCAenzymes were found (see Supplementary Fig. 3 online). The mito-chondrial form of isocitrate dehydrogenase was found in the heav-iest fraction, fraction 1, suggesting that it is part of an extremelylarge complex, and the cytosolic form of malate dehydrogenase wasidentified in the insoluble pellet. However, other TCA enzymes anddehydrogenases were found in lighter fractions 7–10. These resultssuggest that a single supramolecular complex, such as is found inyeast, was not present in our sucrose gradients.

Twelve proteins involved in carbohydrate metabolism were iden-tified, including several isoforms of aldehyde dehydrogenase, andthese were dominated by the mitochondrial form. Notably, alde-

hyde dehydrogenases were found to be an abundant protein familyduring a recent survey of the plant mitochondrial proteome bytwo-dimensional PAGE25. Glycogen phosphorylase and glycerol-3-phosphate dehydrogenase (two isoforms) were identified, and glyc-erol kinase was found in a heavy sucrose fraction (seeSupplementary Fig. 3 online), suggesting an association with amultimeric protein complex.

Because the primary energy source of the heart is oxidation oflong-chain fatty acids, it is not surprising that the enzymes involvedin β-oxidation were well represented, with 48 distinct protein identi-fications. The best coverage was for the α and β subunits of the tri-functional protein. Two isoforms each of monoamine oxidase,aspartate aminotransferase, and methylcrotonoyl–coenzyme A car-boxylase, as well as dihydrolipoamide dehydrogenase (a multifunc-tional subunit in a number of different complexes) had the mostsequence coverage of the 12 identified proteins involved in aminoacid metabolism. Adenylate kinases constituted four of the total ofseven identified proteins that are involved in nucleotide metabolismwith creatine kinase (two isoforms), having the best coverage of theproteins in this class.

Cell death and defense. We collectively categorized apoptotic,detoxifying, immune- and tumor-related proteins under ‘celldeath/defense.’ Programmed cell death-8 (PCD8, also known asapoptosis-inducing factor, AIF) was by far the predominant apoptot-ic protein of the five identified. Bcl2l13 (also known as Bcl-Rambo)and Smac were also identified. The composition of proteins releasedfrom mouse liver mitochondria undergoing the membrane perme-ability transition has been described previously26. Most proteinsidentified in that study were under 80 kDa in molecular weight andthey included many matrix proteins, presumably reflecting the rela-tively nonselective nature of membrane permeabilization. NeitherBax nor a protein homologous to PCD8, nor any of the caspases,were identified in that previous investigation, but lysosomal cathep-sin B and some peroxisomal proteins were found either as contami-nants or as proteins with dual organellar localization26. In our study,we detected cathepsin isoform D as well as several proteins usuallyclassified as peroxisomal that are listed under the ‘protease’ and‘redox’ classes, respectively (see below). Therefore, given that ourmitochondrial preparation was highly purified, this result supports

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Figure 2. Distribution of the 498 functionally classified proteins.Assignments were made on the basis of (i) in-house expertise inmitochondrial biology, (ii) classifications provided in the MITOP3 database,and (iii) information provided in the National Center for BiotechnologyInformation’s Locuslink website (http://www.ncbi.nlm.nih.gov/LocusLink).

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the notion of dual organellar localization for these proteins.Redox proteins. The group of redox proteins included 29 iden-

tifications, the best coverage being for nicotinamide nucleotidetranshydrogenase (two isoforms). Peroxiredoxins (four isoforms),cytochrome B5 reductase (two isoforms), quinone oxidoreductase(NADPH), and mitochondrial superoxide dismutase (SOD2, alsoknown as MnSOD) were also major proteins in this group. Theseresults are consistent with a recent study in which SOD2, peroxire-doxin III, mitochondrial thioredoxin reductase, and mitochondri-al thioredoxin were found as the major components of the humanmitochondrial antioxidant defense system27. We also found glu-tathione peroxidase as a single peptide match. Neither glutathioneperoxidase nor mitochondrial thioredoxin reductase were identi-fied directly in that previous study27, and therefore their detectionhere is especially revealing.

Proteases and protein targeting. Of the 14 proteases identified,the mitochondrial endopeptidase La homolog was most abun-dant. We found 33 proteins in the ‘protein targeting’ category—16of them involved in protein stabilization—which were exemplifiedby molecular chaperones, heat-shock proteins, and 3 cyclophilins(A, B, and F). The human cyclophilin F sequence, more commonlycalled cyclophilin D, has a key role in ischemic cell death28. Weidentified eight subunits of the translocases of the inner and outermitochondrial membrane, TIM and TOM, which function in pro-tein import to mitochondria. The TIM and TOM complexes havebeen extensively studied in lower eukaryotes, including yeast andthe filamentous fungus Neurospora crassa29. Mammalianhomologs of the TIM and TOM proteins are less well character-ized30. The approximate sizes of the TIM and TOM complexes inyeast are known. We can estimate the sizes of these complexes inhumans based on their fractionation in the sucrose gradients, inwhich the OXPHOS complexes serve as size calibration standards(see Supplementary Fig. 3 online). For example, TOMM40 (alsoknown as TOM40) and TOMM22 (also known as TOM22) werefound in sucrose density gradient fraction 7, suggesting that theyare part of an ∼ 100 kDa complex that is similar in size to succinatedehydrogenase (complex II) and much smaller than the 400 kDageneral import/insertion pore (GIP) of yeast31. These observationsare consistent either with partial dissociation of the TIM andTOM complexes or with species-specific differences in subunitcomposition and complex structure. A homolog to the yeastTOM20 was found in the densest sucrose fraction, fraction 1,

along with the very large complexes (for example, ribo-somes) and, interestingly, metaxin-2. Although earliersuggestions that metaxin might be the mammalian coun-terpart of the yeast TOM37 in the TOM complex30 havebeen controversial32, our results suggest that the humanhomolog of the yeast TOM20 and metaxin-2 might associ-ate in an uncharacterized supercomplex.

Nonmitochondrial proteins. We identified 19 distinctcytoplasmic, glycolytic enzymes in our preparation bymass spectrometric analysis. These represent either minorcontaminants, proteins that have physical associationswith mitochondria, or proteins or isoforms that are alsoresident within mitochondria for which a mitochondrialassociation has not been previously established. It is note-worthy that the three isoforms of GAPDH and two iso-forms of aldolase exhibit neutral to basic pI values andmay electrostatically associate with the mitochondrialouter membrane, as has been previously reported33. Wetested this hypothesis by washing mitochondria with 150 mM KCl and found that GAPDH (pI 8.7), aldolase (pI 8.2), and other, nonmitochondrial proteins such asimmunoglobins (pI 8–8.5), albumin (pI 6.3), hemoglobin

β-chain (pI 7.3), and myoglobin (pI 7.9) were released. Such asso-ciations may or may not be biologically relevant. We also identi-fied the major glycolytic enzyme hexokinase, whose associationwith mitochondria has been shown in previous studies to serve aregulatory role. Mitochondrial binding activates hexokinase insome tissues and therefore modulates glycolysis34. Furthermore, ithas recently been shown in human HeLa cells and rat hepatocytesthat a hexokinase-mitochondrial association inhibits Bax-inducedrelease of cytochrome c and thereby regulates apoptosis35. The sig-nificance of the other glycolytic enzymes identified in our highlypurified mitochondrial preparation remains to be determined.

Other apparent contaminants were mainly confined to the n-dodecyl-β-D-maltoside–insoluble material that was not loadedon the sucrose gradient, as reported previously6. Some of these pro-teins may represent artifacts of the isolation procedure, whereasothers may reflect the cytoskeletal architecture with which mito-chondria are intimately associated. For example, it has long beenknown that mitochondrial movement takes place on microtubules36

and tubulin has recently been found to associate with VDAC37.Furthermore, mitochondria localized in the perinuclear region inrat cardiomyocytes provide the ATP required for histone import intothe nucleus38. This intimate association between mitochondria andthe nucleus, coupled with electrostatic effects, may explain the pres-ence of histones in our current and previous preparations6 as well asin those of other investigators39. Given these reports, we draw no con-clusions as to whether the structural and apparent contaminant pro-teins we have found are mitochondrial or mitochondrially associated.

Undetected proteins and dynamic range. Although we haveassembled a list of physiochemically and functionally diverse pro-teins, our choice of methodology may not be optimal for certainproteins. For example, in the case of the oxidative phosphorylationcomplexes, 9% of the unidentified subunits include proteins thateither are very hydrophobic (for example, ATPase subunit C, theproteolipid anchor of complex V, with a hydrophobicity index forthe mature protein approaching 1.0) or are small, with few trypticcleavage sites (for example, NADH dehydrogenase mitochondriallyencoded subunit 4L, with one 23-amino acid tryptic peptide).Similarly, the absence of other proteins may reflect either physicalproperties or chemistries not conducive to detection by our analyt-ical strategy, or their low abundance in a complex mixture.

To date, quantitation of protein copy number in mitochondria,or cells in general, has been limited. However, there is recent data

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Figure 3. Recombinant LETM1 localizes to mitochondria. COS-1 cells weretransiently transfected with a LETM1-FLAG cDNA for 24 h, incubated withMitotracker Red for 30 min, fixed and immunostained with an anti-FLAG antibody.(A) Anti-FLAG immunostaining of recombinant LETM1. (B) Mitotracker Red.(C) Overlay of FLAG and Mitotracker Red. The LETM1 cDNA was cloned by RT-PCRusing the gene-specific primer sequence (5′-CTAGCTCTTCACCTCTGCGAC-3′)from human heart poly(A)+ RNA (BD Biosciences Clontech, Palo Alto, CA) andligated into the mammalian expression vector pcDNA3.1+ (Invitrogen, Carlsbad, CA).The 3′ PCR oligonucleotide incorporated a FLAG epitope tag. COS-1 cells weretransiently transfected with the LETM1/pcDNA3.1+ plasmid using the Fugenetransfection reagent (Roche Applied Science, Indianapolis, IN). After 24 h oftransfection, the cells were loaded with Mitotracker Red (Molecular Probes, Eugene,OR), washed, and fixed with 3% paraformaldehyde. Immunofluorescence stainingwas performed using the M2 anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO) andthe Alexa-488 goat anti-mouse secondary antibody (Molecular Probes).

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on the relative amounts of the respiratory-chain complex III andthe pyruvate dehydrogenase complex (PDH) in human heartmitochondria, which are found in a ratio of 200:1 (ref. 40). Amongthe polypeptides identified in our study is the E3 PDH bindingprotein, which is present in 12 copies per complex—that is, in a17-fold lower copy number than subunits of complex III. Less rig-orous evidence that polypeptides in much lower copy numberthan PDH are present comes from the significant hit rate for subunits of mitochondrial ribosomes and the protein importmachinery, all of which are present in low abundance22,41. Thus ourdatabase contains proteins of wide-ranging copy number.

Unclassified and unknown proteins. About 20% of all the pro-teins identified here have not been assigned to a functional class.These include dominant proteins such as mitofilin (two isoforms),leucine-rich proteins containing a PPR-motif, CGI (comparativegene identification) proteins, and 71 proteins for which theGenBank annotation was listed as ‘hypothetical protein’ or‘unknown’. All of these protein sequences were subjected to HMMmotif searches against the Pfam database10 (the distribution of thetop scoring motifs for 57 of the proteins can be found on theauthors’ website; see URL). The remainder had no clearly recog-nizable motif. Some motifs have a clear functional implication(for example, ‘AMP-binding enzyme’), whereas others are morecryptic (for example, ‘domain of unknown function DUF20’). Forthese proteins, as well as for the known proteins that are normallyassociated with other cellular sites, it will be necessary to validatethe intracellular localization.

Recent high-throughput immunolocalization of 2,744 taggedgene products in yeast42 provides some insight into the subcellulardistribution of our unknown proteins. We performed basic localalignment search tool (BLAST) searches on our entire list ofunknown proteins and found 24 sequences that had high homolo-gy to yeast proteins with Expect values less than 1 × 10-10 (seeSupplementary Table 4 online). Of these yeast homologs, 2 hadmitochondrial annotations and a further 15 were successfullyimmunolocalized in the high-throughput study42. Seven of theselatter proteins were found in mitochondria and eight were foundin other subcellular compartments and the cytoplasm.

Although it is difficult to extrapolate this trend across phyla tohumans, we have begun validation experiments on selected pro-teins that we have identified but that were not previously knownto have a mitochondrial localization. Initially, we studied a RABprotein, RAB11 (see Supplementary Table 2 online), because thisclass of proteins is suggested to comprise organelle-specificGTPases43 and an antibody was commercially available. RAB11was significantly enriched in metrizamide-purified human heartmitochondria by western blot (see Supplementary Fig. 1 online).We have also investigated LETM1, a leucine-zipper-EF-hand–containing putative integral plasma membrane protein, whichcontains a calcium-binding motif. Deletions in this protein areassociated with Wolf-Hirschhorn syndrome (WHS)44. Notably, theprotein shows high homology to a Drosophila melanogaster pro-tein that was localized to mitochondria45. As no commercial anti-bodies specific for this protein are available, we cloned the LETM1cDNA with a C-terminal FLAG epitope tag for visualization.Western blot analysis of COS-1 cells that were transiently trans-fected with this construct revealed a band of the correct molecular weight (data not shown). When the subcellular localiza-tion was examined by immunofluorescence, the LETM1-FLAG

construct co-localized with Mitotracker Red, a mitochondrialmarker (Fig. 3).

Conclusions. We have established a reference database of 615mitochondrial and mitochondrial-associated proteins of thehuman heart. This database currently provides the most compre-hensive overview of the mitochondrial proteome in terms of theproteins’ physiochemical properties as well as their functionalclassification. By rigorously purifying mitochondria to minimizeextraorganellar contamination, we have produced evidence for amitochondrial association of proteins that are normally found atother cellular locations. We have also found many potentially newmitochondrial proteins that have previously only been predictedthrough gene-finding programs. By using a methodology thatlargely preserves multimeric protein complexes, we have identifiedprotein-protein associations that can be validated through the useof protein-protein interaction databases and immunocapturestrategies46. We are currently employing immunolocalization,cloning, and expression strategies on subsets of proteins to gain adeeper understanding of their functional biology in the mitochon-drion. Ultimately, a comprehensive, annotated mitochondrial pro-teome database will be of immense utility for identification andvalidation of targets for therapeutic treatment of human diseases.

Experimental protocolHuman heart mitochondria, isolated by differential centrifugation fromthree donor hearts, were obtained from Analytical Biological Services(Wilmington, DE). The donors were between 16 and 64 years of age andshowed no indication of cardiovascular disease. Mitochondria (40 mgtotal) were further purified by metrizamide gradient centrifugation47 andtheir integrity and purity was assessed by western analysis (seeSupplementary Fig. 1 online).

The purified mitochondria were solubilized with n-dodecyl-β-D-maltosideand the complexes partially resolved on sucrose gradients as described previ-ously5,6. The gradient fractions were collected, concentrated, and quantifiedas described previously6. Electrophoresis, gel processing, and in-gel diges-tion conditions are described in detail in the Supplementary experimentalprotocol online. Automated target spotting and data acquisition using aVoyager DE-STR matrix assisted time of flight (MALDI-TOF) mass spec-trometer were performed using Proteomics Solution 1 (Applied Biosystems,Foster City, CA) as described previously6. Automated LC/MS/MS analysisusing a MicroTech Ultimate LC system coupled to a Finnigan LCQ DECAion-trap mass spectrometer equipped with a Finnigan dynamic nanospraysource was performed as described previously48. The identification of mito-chondrial proteins was based upon PMF using the program ProteinProspector49 and upon LC/MS/MS using two independent searching algo-rithms, SEQUEST50 and SonarMSMS51. A full description of the scoring cri-teria and the process of data integration is provided in the Supplementaryexperimental protocol online.

URL. For additional data, see http://www.mitokor.com/files/

Note: Supplementary information is available on the Nature Biotechnologywebsite.

AcknowledgmentsThe authors thank Neil Howell and Christen Anderson for helpful comments,and Paul Haynes and Ross Hoffman for technical advice.

Competing interests statementThe authors declare competing financial interests: see the Nature Biotechnologywebsite (http://www.nature.com/naturebiotechnology) for details.

Received 11 October 2002; accepted 14 January 2003

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