expanded coverage of the human heart mitochondrial proteome using multidimensional liquid...

Expanded Coverage of the Human Heart Mitochondrial Proteome

Using Multidimensional Liquid Chromatography Coupled with

Tandem Mass Spectrometry

Sara P. Gaucher,†,§ Steven W. Taylor,‡,| Eoin Fahy,‡,⊥ Bing Zhang,‡,# Dale E. Warnock,‡,¶

Soumitra S. Ghosh,‡,| and Bradford W. Gibson*,†

Buck Institute for Age Research, Novato, California 94945, and MitoKor, San Diego, California 92121

Received November 5, 2003

Recent evidence suggests that mitochondria are closely linked with the aging process and degenerativedisorders such as Alzheimer’s disease and Parkinson’s disease. Thus, there has been increasing interestin cataloging mitochondrial proteomes to identify potential diagnostic and therapeutic targets. We havepreviously reported results of a one-dimensional electrophoresis/liquid chromatography MS/MS studyto characterize the proteome of normal human heart mitochondria (Taylor et al. Nat. Biotechnol. 2003,21, 281-286). We now report two subsequent studies where multidimensional liquid chromatographyMS/MS was investigated as an alternative means for characterizing the same sample.

Keywords: mitochondria • LC/MS/MS • MudPIT • MDLC

Introduction

Mitochondria are the powerhouses of cells and also play anintegral role in ion homeostasis, fatty acid oxidation, intracel-lular signaling and in the regulation of oxidative stress and celldeath processes.1 Furthermore, recent evidence suggests thatmitochondria are closely linked to the aging process and tomany degenerative disorders such as Alzheimer’s disease,2

Parkinson’s disease3,4 and diabetes mellitus.5 The emerging roleof mitochondrial dysfunction in disease has led to a surge ofinterest in studying mitochondrial proteomes6-17 to identifypotential diagnostic and therapeutic targets. The exact numberof mitochondrial proteins is not known, but is estimated to beon the order of 1000 proteins.18 Only 13 of these proteins areencoded by the mtDNA; the remainder is encoded by thenuclear genome and must be imported into the mitochondria.19

Temporal variation in protein expression (i.e., during respira-tion, biogenesis, apoptosis, etc) and tissue distribution andlocalization to mitochondrial sub-compartments (mitochon-drial inner membrane, matrix, intermembrane space, etc)increases the complexity of the analysis.

The method of choice for proteomic studies to date has beenbased primarily on two-dimensional electrophoresis (2DE) andeither peptide mass fingerprinting (PMF) or LC/MS/MS analysis

of the peptides generated by in-gel digest of excised spots.However, the mitochondrial proteome is particularly recalci-trant to this type of analysis.11 A large percentage of theseproteins are integral or peripheral membrane proteins becausemany of this organelle’s functions occur within its inner andouter membranes. Such proteins are quite difficult to solubilizeand resolve well by 2DE due to their extremely hydrophobiccharacter. In addition, many mitochondrial proteins are small(MW < 15 kD) and basic (pI > 9).20 To improve the coverageof these protein classes, a series of gels may be run with varyingpH gradients for the isoelectric focusing step. Previous studieson human mitochondrial proteomes with the sample analyzedon a single 2DE gel resolved up to ca. 1500 spots of which 50-60 unique proteins were identified.6,9 Improvements in proteincoverage were obtained on the mitochondrial proteomes ofhuman placenta14 and rat liver7,10 by running a series of gelsobtained from aliquots of the sample enriched for variousprotein classes (∼100 gene products identified),7 or from thesame sample by varying the first dimension pH gradient (∼130and ∼200 gene products identified).10,14 However, despite theimprovements in the total number of proteins identified, thelow number of basic proteins and membrane proteins identi-fied remained a methodological issue.

Two studies of mitochondrial proteomes have reported theuse of alternate separation strategies. Pflieger et al.11 identified179 gene products from yeast mitochondria using one-dimensional electrophoresis/liquid chromatography MS/MS(1DE/LC/MS/MS). Their results are comparable in number withthe six 2DE gels run by Fountoulakis et al.10 but encompass amuch greater percentage of small, basic, and membraneproteins. Spahr et al.8 bypassed gels altogether to study theproteins released from mouse liver mitochondria undergoingatractyloside-induced membrane permeabilization. They iden-

* To whom correspondence should be addressed. 8001 Redwood Blvd.,Novato, CA 94945. Phone (415) 209-2032. Fax (415) 209-2231. [email protected].

† Buck Institute for Age Research.‡ MitoKor.§ Currently at Sandia National Laboratories, Livermore, CA 94551.| Currently at Amylin Pharmaceuticals, San Diego, CA 92121.⊥ Currently at San Diego Supercomputer Center, University of California

San Diego, La Jolla, CA 92093.# Currently at Department of Breast Medical Oncology, University of Texas

M. D. Anderson Cancer Center, Houston, TX 77030.¶ Currently at Neose Technologies, San Diego, CA 92121.

10.1021/pr034102a CCC: $27.50 2004 American Chemical Society Journal of Proteome Research 2004, 3, 495-505 495Published on Web 04/02/2004

tified 108 gene products after only nine LC/MS/MS experi-ments, and the experimental design was conducive to high-throughput studies. Three recent large scale studies onnonhuman mitochondrial proteomes, yeast,15 mouse,16 andplant17 have generated lists of 750, 591, and 416 mitochondrialproteins for these organisms, respectively. The strategy for thesestudies included pooling data from multiple types of experi-ments or performing experiments on different tissues to expandupon the lists of proteins that could be generated from anyone of these experiments alone.

The long term objective of our current studies is to com-prehensively catalog the mitochondrial proteome from humanheart. Given the improved proteome coverage using 1DE/LC/MS/MS and nongel based approaches, we applied thesetechniques to a highly purified mitochondria preparation.Recently, the results of our analysis using 1DE/LC/MS/MS werepresented.13 In this report, we describe the use of multidimen-sional liquid chromatography coupled with tandem massspectrometry (MDLC/MS/MS) toward the mapping of themitochondrial proteome.

Experimental Section

Materials. Mitochondria were obtained from human heart(Analytical Biological Services, Wilmington, DE) and furtherpurified using a Percoll/metrizamide gradient as describedelsewhere.9 Samples for each of the two MDLC experimentsdescribed below (Expt 1 and Expt 2) were from separate poolsof donors: Sample for Expt 1 was isolated from the same poolof three donor hearts as described previously,13 and sample forExpt 2 was isolated from a different pool of two hearts. Thelatter donors were both approximately 45 years of age, and thecause of death was unrelated to cardiovascular disease. Allchemical reagents used were of analytical grade or better.

MDLC/MS/MS Analysis 1 (Expt 1). In these experiments, aportion of the protein fractions generated for the 1DE/LC/MS/MS analysis13 was used. The fractions had been obtained bysolubilizing purified human heart mitochondria in n-dodecyl-â-D-maltoside buffer and separating the proteins, includingintact complexes, using sucrose density centrifugation.12,20 Tensucrose gradient fractions (labeled SG1 through SG10) and oneinsoluble pellet (labeled LMP, for lauryl maltoside pellet) wereused for Expt 1. Typically, 100 µg of each sample was dissolvedin 100 µL of 50 mM Tris/0.1% SDS, pH 8.5. A 2 µL portion of50 mM TCEP solution was added, and the sample was boiledfor 10 min. The sample was then incubated in the dark at RTfor 1 h after adding 5 µL of 100 mM iodoacetamide. Trypsinwas added (100 µL of a 0.1 µg/µL solution in water), and thesample was incubated at 37 °C overnight.

A portion of each digest (equivalent to 50 µg protein) wasdiluted with 2 mL of 2 mM KH2PO4 in 25% aqueous acetonitrile(pH < 3) and applied manually to a strong cation exchange(SCX) cartridge (POROS 50 HS, 4.0 mm × 1.5 cm, AppliedBiosystems, Foster City, CA) fitted with a syringe adapter. Afterloading, peptides in fractions SG1-SG10 were eluted withsuccessive salt steps of 100 µL (0.5 column volumes) each of35, 52.5, 70, 87.5, 105, 122.5, 140, 157.5, 175, and 350 (4×) mMKCl in 10 mM KH2PO4 buffer (pH < 3) containing 25% aqueousacetonitrile. Fractions were collected in 100 µL increments, andpeptides were contained in fractions 3-12 (corresponding tothe solvent fronts of each of the 10 salt steps). An alternateelution series was initially tried using LMP and SG10, wheresuccessive salt steps of 500 µL each (2.5 column volumes) of52.5, 87.5, 140, 175, and 350 (2×) mM KCl were used to elute

the peptides, and fractions were collected in 250 µL increments.However, this method introduced too much salt into thefractions, resulting in relatively poor binding to the C18 pre-column during LC/MS/MS and prompted the switch to theimproved elution series for SG1-SG10. Data collected on SG10with both methods was included in the final analysis. SampleLMP was not refractionated using the final elution series dueto sample constraints.

In addition to fractionating the eleven samples (SG1-SG10,LMP) as described above, a 50 µg portion of SG3 and of LMPwere treated by using the SCX cartridge only as a clean up stepto remove SDS and trypsin from the digest. In this case,peptides were eluted in a single step using 350 mM KCl.

RP-HPLC was performed with an Ultimate Nano LC System(Dionex, Sunnyvale, CA). Samples (2.5-10 µL, estimated 0.3-2.0 µg total) were loaded at 20 µL/min with 0.05% formic acidonto a C18 precolumn - either 0.3 × 1 mm (LC Packings/Dionex) or 0.5 × 2 mm (Michrom, Auburn, CA) - for cleanup/concentration. After washing for 10 min, peptides were back-flushed onto a 75 µm × 15 cm nanocolumn - 3 µm × 100 ÅC18 (LC Packings/Dionex) or 5 µm 300 Å C18 (Vydac, Hesperia,CA) and eluted with an acetonitrile gradient, typically 5-27%B in 75 min then 27-50% B in 10 min (solvent A ) 2% aqueousacetonitrile + 0.05% formic acid, solvent B ) 98% aqueousacetonitrile + 0.05% formic acid). Spectra were acquired onan Applied Biosystems/MDS QStar. Information DependentAcquisition (IDA) was performed using the following param-eters: 1 s MS; 3 s MS/MS on 2+ or 3+ ions at 400-1200 m/z, 5cps minimum intensity, 60-120 s dynamic exclusion. All MS/MS spectra recorded for these samples were used in thesubsequent data analysis.

Additional IDA experiments were performed on the unfrac-tionated portions of SG3 and LMP. LMP (0.75-1.0 µg) wasloaded and eluted as described above; IDA was carried out asdescribed above but varying the m/z range used for precursorion selection. The following ranges were used: m/z 400-1200,350-460, 450-560, 550-660, 650-760, 750-860, 850-1200. ForSG3, LC/MS/MS was performed (as for the SCX separatedfractions) on a 0.75 µg and a 3.0 µg portion of the unfraction-ated sample. These two experiments were then repeated usinga precursor ion exclusion list based on all species selected forMS/MS in the LC/MS/MS experiment on 0.75 µg of sample.Finally, IDA was carried out on 3.0 µg portions of this sampleusing two narrow m/z ranges for precursor ion selection, m/z550-650 and 650-750. All data acquired was included in thefinal analysis.

Expt 1 Data Interpretation. Sonar MS/MS21 (GenomicSolutions, Ann Arbor, MI) was used to search the spectraagainst the human subset of the NCBInr protein database.Search parameters were as follows: precursor ion tolerance )( 0.2 Da, product ion tolerance ( 0.2 Da, enzymatic cleavage) trypsin, allowed missed cleavages ) 1, fixed modification )carbamidomethylation (Cys), variable modification ) oxidation(Met). Matches with a protein expectation value of e10-3 (99.9%confidence) were automatically accepted. Matches with aprotein expectation value e1 and g10-3, and a peptideexpectation value e1 were manually inspected.22 The resultinglist of 253 proteins obtained from this analysis was less thanhalf that found previously by 1DE/LC/MS/MS. It was alsoobserved that several proteins known to be present but likelyto be supported by only one or two peptide sequences wereabsent from the list. Because protein expression values calcu-lated by Sonar depend on the total number of spectra submit-

research articles Gaucher et al.

496 Journal of Proteome Research • Vol. 3, No. 3, 2004

ted for a search21 we surmised that proteins identified by onlya single spectrum in such a large number of submitted spectrawould not have a “significant” expectation value and wouldgo unreported.

The same data set was therefore submitted to Mascot MS/MS Ions Search23 (Matrix Science, London) using the humansubset of the NCBInr protein database and search parametersanalogous to those listed above. Peptide matches with a scoreof 33 or greater (95% confidence) and corresponding to proteinidentifications not found using Sonar MSMS were manuallyinspected and if accepted, merged with the list of peptidesidentified by Sonar.

MDLC/MS/MS Analysis 2 (Expt 2). This mode of MDLC wasessentially the same as described by Washburn et al.24 withslight modification. Mixed bed columns were constructed bysequentially packing a 75 µm internal diameter PicoFrit (NewObjective, Woburn, MA) with 10-15 cm of C18 (Michrom Magic)followed by 3-5 cm of SCX resin (PolySULFOETHYL Asparta-mide, PolyLC, Columbia, MD) by means of a helium pressurecell (Brechbuehler, Spring, TX). Metrizamide-purified mito-chondria were solubilized in 8 M urea; the resulting superna-tant was labeled the “soluble fraction” and the remainingmaterial was labeled the “insoluble fraction.” The insolublefraction was subject to CNBr digestion in 90% formic acid. Afteran overnight incubation at room temperature in the dark, thesolution was adjusted to pH 8.5 by addition of solid ammoniumbicarbonate (NH4HCO3) taking care to avoid losses duringfrothing. After dilution of the soluble fraction to 2 M urea with0.1 M NH4HCO3 and dilution of the insoluble fraction with asimilar quantity of MilliQ water, the samples were treatedidentically. DTT was added to a final concentration of 1 mMand incubated for 1 h at 54 °C after which iodoacetamide wasadded to a final concentration of 10 mM and incubated for 30min in the dark. Endoprotease Lys C (Wako) was added as 0.23U of activity to 1 mg protein, and the resulting mixture wasincubated at 37 °C (pH 8.5) for 24 h. (Protein content wascolorimetrically assayed using Biorad DC, detergent compatiblereagent, in the initial soluble and insoluble fractions which were1.66 mg and 1.87 mg, respectively.) The mitochondrial proteinsfrom each preparation were then aliquoted into 1.5 mLEppendorf tubes and further digested with immobilized trypsin(Poroszyme, Applied Biosystems) in 70 mM NH4HCO3, 5%acetonitrile, 1mM CaCl2 with agitation for 48 h at 37 °C. Aftercentrifugation, the beads were discarded and the digestscleaned up and concentrated using C18 solid-phase extractioncartridges (SPEC-Plus PT C18, Anysys Diagnostics, Lake Forest,CA) per the manufacturer’s instructions. Finally, the digestswere concentrated from 200 µL to less than 10 µL using aspeedvac, 50 µL of solvent A (95% water, 5% acetonitrile, 0.2%formic acid) was added, and the digests were stored at -80 °Cuntil use. For MDLC/MS/MS analysis, digests were loadeddirectly onto the mixed bed Picofrit column previously equili-brated with solvent A by means of a helium pressure cell at1000 PSI that corresponded to a flow rate of approximately 300nL/min. Approximately 200 µg total peptide digest in 5-10 µLvolume was loaded, an overestimate corresponding to theinitial protein determination assuming total recovery from allsteps. After the samples were loaded, the PicoFrit column wasinserted into a simple nanospray source based on that de-scribed in the literature25 (constructed by the University ofCalifornia, San Diego Department of Chemistry and Biochem-istry fabrication facility), and connected to the liquid junctionand capillary LC via an Upchurch Cross fitting, which also

serves as a flow splitter. The Micro-Tech Ultra Plus II ProteomicSystem (Vista, CA) consists of 2 binary pumping systemsconfigured to pump from 4 solvent reservoirs, A (95% water,5% acetonitrile, 0.2% formic acid) and B (80% acetonitrile, 20%water, 0.2% formic acid), and C (500 mM ammonium acetate,5% acetonitrile, 0.2% formic acid) and D ) A. The flow ratewas adjusted so that after splitting it was typically 400-700 nL/min (20-25 µL per minute presplit). The gradients were similarto the fully automated chromatography runs described in theliterature.24,25 The first step of 117 min consisted of an 80 mingradient from 1 to 80% buffer B, followed by 10 min at 80%and then reequilibration with solvent A. The next steps were124 min each with the following profile: 3 min of x% buffer C,10 min of 99% buffer A, a 90 min gradient from 0 to 60% bufferB which was held at 60% for 10 min followed by reequilibrationwith solvent A. The 3 min buffer C percentages (x%, above)used in steps 2-13 were as follows: 2, 5, 7, 10, 20, 30, 40, 50,60, 70, 80, 90, and 100%. On 2 runs from the insoluble fractionsome spraying problems were experienced, and so theseexperiments were accelerated by using only 75% and 100%steps after the 50% step. For the final salt step, the reservoircontaining buffer C was replaced with 1 M ammonium acetate/5% acetonitrile/0.2% formic acid and the column was typicallywashed for 15 min to remove strongly bound peptides fromthe SCX resin. The 90 min gradient (0 to 60% B) was then runas above. Finally, a 30 min wash with solvent A followed by agradient from 1 to 80% buffer B over 80 min, held at 80% B for10 min, then at 100% B for 20 min, was used to elute theremaining peptides from the column. Mass spectra wereacquired on a Finnigan LCQ DECA ion trap mass spectrometerin the m/z 400-1400 range. After one full scan MS of thecolumn effluent was recorded, three MS/MS spectra of the thirdmost, second most and most intense MS peaks were sequen-tially recorded over the m/z 100-2000 range with an isolationwidth of 2.7 and normalized collision energy setting of 35.Dynamic exclusion was employed to select the maximumnumber of unique peptide peaks from the chromatograms.After replicate MS/MS spectra were acquired for a precursorion, the m/z value of the ion was placed on an exclusion listfor 7 min.

Expt 2 Data Interpretation. Each chromatogram was sub-sequently analyzed with the program SEQUEST26 using thehuman subset of the NCBInr protein database (21 May, 2003)that had been “indexed for speed” with carbamidomethylationas a static modification of cysteine (+57.0 Da) using TurboSE-QUEST software in Bioworks 3.1(ThermoFinnigan). For thesoluble fraction indexing also specified oxidation of methionine(+16 Da) and carbamylation of lysine (+43 Da, includedbecause urea was used for solubilizing) as differential modifica-tions. For the insoluble fraction conversion of methionine tohomoserine in the presence of cyanogen bromide was specifiedas a static modification (-30 Da) and unmodified methionine(+30 Da) and oxidation of tryptophan (+16 Da) were specifiedas differential modifications with the enzyme editor adjustedto specify methionine as well as arginine and lysine as cleavagesites.

A set of web-based batch programs, written in Perl, weredesigned to enable automated raw data preparation, searchingand reporting of SEQUEST runs. All peptide matches werefiltered based on their SEQUEST cross-correlation (Xcorr)values and ∆corr values (a measure of the difference in Xcorrbetween the best and next best peptide match). Peptidematches with ∆corr values g0.1 and Xcorr values g1.7 (Charge

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Journal of Proteome Research • Vol. 3, No. 3, 2004 497

State 1) or g2.0 (Charge State 2) or g3.0 (Charge State 3) weretentatively accepted.

Spectral data from each of the LC/MS/MS runs were alsosearched against the same database (NCBInr, human subset,May 2003 release) using the SonarMSMS algorithm running onLinux. Spectral data were first merged into a set of input files,and thus processed in batch mode, using a web-client emula-tion program written in Perl. Peptide matches with a PeptideExpect value of e0.01 and Protein Expect value of <1 for thecorresponding protein were tentatively accepted.

Finally, matches for peptide sequences obtained from theSEQUEST or SonarMSMS searches that corresponded to pro-teins not identified previously by 1DE/LC/MS/MS were manu-ally inspected before inclusion in the final list of identifiedproteins.

Data Integration. A final round of curation was performedon each of the three sets of Genbank IDs identified by 1DE/LC/MS/MS and MDLC/MS/MS (Expt 1 and Expt 2) methods.This consisted of first filtering our results with the freelyavailable program, CD-HIT.27 All protein accession numbersidentified as the top hit from the database search on each MS/MS spectrum were used to create a FASTA-formatted proteindatabase file. CD-HIT was then used to group those accessionnumbers having greater than 90% protein sequence identity.Members belonging to each cluster were then analyzed todetermine whether there was supporting LC/MS/MS evidencefor unique peptide(s) for each sequence. Oracle database tableswere constructed containing all possible tryptic peptides forproteins in the human subset of GenBank and all trypticpeptides detected in LC/MS/MS searches which exceeded thethreshold limits. A web-based query program written in-houseenabled computation of unique tryptic peptides correspondingto any 2 protein identifications of interest (isoforms, splice-variants etc). This program also determined which, if any, ofthese unique tryptic peptides were detected in LC/MS/MSsearches. Where no MS/MS data was available to conclusivelysupport the presence of more than one member of a particularcluster, all accession numbers within the group were replacedby a single number and the annotations were concatenated.Thus, this software tool was used to efficiently interrogateproteins with greater than 90% sequence identity and eliminateredundant sequences. In the case of the 615 protein sequencesoriginally found by 1DE/LC/MS/MS this procedure identified42 IDs with 90% or more sequence homology to another entryin this list, but without MS/MS data to distinguish betweenthem. Thus, the final protein tally has been reduced by thisnumber in the subsequent discussion to reflect only uniqueproteins identified.

Results and Discussion

Sample Preparation. Two separate MDLC/MS/MS analysesof human heart mitochondrial proteins were performed andare referred to as “Expt 1” and “Expt 2”. In Expt 1, a portion ofthe sample previously analyzed by 1DE/LC/MS/MS was used.Here, the entire mixture of mitochondrial proteins had beenfractionated into 12 pools (11 fractions of soluble protein plus1 insoluble protein pellet) based on sucrose density gradientcentrifugation (SDGC).12,20 This SDGC method tends to keepprotein interactions intact and thus separates on the basis ofprotein complex size. For example, each of the electrontransport chain complexes (ranging in size from ∼150-900kDa) was separated into different fractions as measured byWestern blot.12 The different protein profiles in each fraction

resulting from the separation was demonstrated by SDS-PAGE.13 Ten of the soluble protein pools and the insolubleprotein pellet were digested with trypsin. The resulting peptidemixtures were subfractionated using a strong cation exchangecartridge fitted with a syringe adaptor. Selected strong cationexchange peptide subfractions were then analyzed by reversephase LC/MS/MS, and over 46 000 MS/MS spectra wererecorded.

A tryptic digest of bovine serum albumin (BSA) was used totest the efficacy of the strong cation exchange cartridge. MALDIspectra were acquired on the fractions collected from salt stepelution. Spectra from three of these fractions are given in Figure1. Two fractions from consecutive salt step elutions (fractions4 and 5) are shown along with one noncontiguous step (fraction9). There is some overlap between the peptides eluted fromneighboring salt steps, although the relative abundances ofthese common peaks are quite different. Furthermore, peptidesunique to each of these fractions are still observed. There isalmost no overlap between fractions 5 and 9. Thus, overall,different peptide profiles are contained in each fraction. Wealso found that this strong cation fractionation procedureproduced consistent results: There was close correspondenceof the majority of peaks between spectra eluted in the samesalt fraction from independent separations of the BSA digest(Supplementary Figure 1). Thus, a manual cartridge-basedseparation strategy appeared to be a satisfactory and conve-nient method of fractionating the human heart mitochondrialprotein digests.

For the second MDLC/MS/MS experiment (Expt 2), an entiremitochondrial protein mixture was separated into two fractions,soluble and insoluble protein. Aliquots of each fraction weresolubilized with the SDS-containing sample buffer and an SDS-PAGE gel was run (shown in Figure 2) demonstrating thedifferent protein profiles contained in each fraction. Eachcomplex peptide mixture resulting from digestion of the twoprotein fractions was loaded onto the head of a biphasiccolumn and subfractionated by strong cation exchange againusing salt steps. The eluent from each salt step was analyzeddirectly, however, by reverse phase LC/MS/MS using the C18

column bed packed in series with the strong cation exchangecolumn bed. On the order of 80 000 MS/MS spectra wererecorded during this analysis.

Data Analysis and Protein Identification. A summary of thenumber of proteins identified in all three studies performedto date is shown in Figure 3. A total of 366 proteins have nowbeen identified by two or more independent studies, 135 ofwhich have been identified in all three studies, thus lendingincreased confidence to these identifications. Both MDLC/MS/MS studies yielded approximately the same number of iden-tifications (314 and 294 for Expt 1 and Expt 2, respectively).This is at least a 2-fold improvement over previous 2DE basedstudies on human mitochondrial proteomes.6,9,14 It is notewor-thy that more total proteins were identified by our initial 1DE/LC/MS/MS study after clustering with CD-HIT (573) than evenfrom the two MDLC/MS/MS studies combined (463). Never-theless, both MDLC-based experiments had value because theyboth identified proteins not found in the previous study (107total additional proteins, 27 unique to Expt 1 and 70 unique toExpt 2). In addition, it is likely that some of these proteins couldonly have been identified by a non gel-based method becausethey were either extremely small or hydrophobic as detailedin the following section. A summary of the 107 proteinsidentified in these mitochondrial samples not reported previ-



ously is given in Table 1. Some of these entries representisoforms of previously identified proteins. Others (for example,Complex V subunit c, NADH dehydrogenase III, and SLAM)represent known mitochondrial proteins not identified in theprevious study. Further information such as specific peptidesidentified, in which experiment, with which algorithm andmatch score is given in Supplementary Tables 1a and 1b.

Because this effort is part of a larger project to generate amitochondrial protein database for public use,28 proteinsreported here have been added to the MitoProteome database(http://www.mitoproteome.org). This database is dynamic andconstantly evolving as new information is obtained regardingthe identified proteins. For example, this list, particularly theproteins of unknown function and proteins not previouslyidentified to associate with the mitochondria, still requiresvalidation on two levels. Manual inspection and/or highsequence match scores only ensure that the data and theproposed sequence assignment are reasonably consistent. Byitself it does not guarantee that a particular assigned sequence(and therefore the assigned protein) is correct. We are thereforecontinuing our protein identification validation process ofcomparing an experimental MS/MS spectrum of interest withthat of the synthetic analogue for the proposed sequence.13,29

The list of mitochondrial proteins is then curated based on theresults of these experiments, to reflect the most up-to-dateinformation.

General Characteristics of Identified Proteins. Proteinchemistry is fundamentally more complex than the chemistryassociated with DNA. Characteristics of acidity, basicity, hy-drophobicity, and size vary widely across any given organellarproteome. Coupled with this is the orders of magnitudedifference in the level of abundance within the mixture and

Figure 1. MALDI spectra from a manual strong cation exchange fractionation of a BSA digest show that different peptide profiles elutein each fraction.

Figure 2. SDS-PAGE gel of aliquots of the two mitochondrialprotein fractions analyzed by MDLC/MS/MS Expt 2. Lane A )molecular weight marker; lane B ) urea soluble fraction; laneC ) urea insoluble protein pellet.

Figure 3. Venn diagram depicting proteins identified by each ofthree analyses. A total of 680 unique proteins were identified.



Table 1. Additional Proteins Identified in Human Heart Mitochondria Using Nongel Based Methods

genbank ID annotation

24485 A1S9 protein (AA 1-803) ubiquitin-activating enzyme E130158852 actin CA15 - sea squirt (Styela clava) (fragments) similar to1354222 aldehyde dehydrogenase E3′17402879 R-2 type VI collagen, isoform 2C2a precursor17149811 R-3 type VI collagen, isoform 5 precursor; collagen VI, R-3 polypeptide2661039 R-enolase10947054 ankyrin 2 isoform 2; ankyrin-2, nonerythrocytic; ankyrin-B21068652 apolipoprotein A-I binding protein4502301 ATP synthase, H+ transporting, mitochondrial F0 complex, subunit c (subunit 9) isoform 34502277 ATPase, Na+/K+ transporting, â-1 polypeptide; ATPase, Na+K+ transporting, â-1 polypeptide27924394 azurocidin 1 (cationic antimicrobial protein 37) Similar to18481635 â I spectrin form â I σ 327462180 â-spectrin 2 isoform 24757862 biphenyl hydrolase-like; Bph-rp; breast epithelial mucin-associated antigen8176547 branched-chain R-keto acid dehydrogenase E1 R subunit4757900 calreticulin precursor; Sicca syndrome antigen A4502517 carbonic anhydrase I4557395 carbonic anhydrase II9651188 cardiac myosin light chain-12134870 carnitine O-palmitoyltransferase (EC 2.3.1.21) I, muscle type - human4557419 CD36 antigen (collagen type I receptor, thrombospondin receptor); CD36 antigen (collagen type I)337765 cerebroside sulfate activator protein15928808 ceroid-lipofuscinosis, neuronal 2, late infantile (Jansky-Bielschowsky disease)4929597 CGI-64 protein [Homo sapiens]27923741 Chaperone-activity of bc1 complex-like, mitochondrial precursor (Chaperone-ABC1-like)5453603 chaperonin containing TCP1, subunit 2 (â); chaperonin containing t-complex polypeptide 1, â subunit4502603 chromobox homologue 4; chromobox homologue 4 (Drosophila Pc class)1244508 clathrin assembly protein 50 - human >gi|1244508|gb|AAA93254.1| assembly protein 504758012 clathrin heavy chain; clathrin, heavy polypeptide-like 29257202 clathrin, heavy polypeptide-like 1 isoform b30851190 Collagen, type VI, R-1 precursor7512358 COX17 homolog, cytochrome c oxidase assembly protein; human homologue of yeast mitochondrial

copper recruitment gene3646132 cysteine desulfurase; putative tRNA splicing protein18999392 cytochrome c oxidase subunit Va4885149 cytochrome c oxidase subunit VIa polypeptide 266257 cytochrome-c oxidase (EC 1.9.3.1) chain III - human mitochondrion23821029 D-lactate dehydrogenase [Homo sapiens] >gi|23506788|gb|AAM50322.1| D-lactate dehydrogenase [Homo sapiens]627367 desmoyokin - human (fragments)30582727 dihydropyrimidinase-like 214149825 dimerization cofactor of hepatocyte nuclear factor 1 (HNF1) from muscle27677886 dJ127D3.2 (Flavin-containing Monooxygenase family protein) [Rattus norvegicus] Similar to27479619 dynein, cytoplasmic, heavy chain 19966867 eIF-5A2 protein; eIF5AII30149784 elongation factor 1 R Similar to31397 fibronectin precursor27478724 FLJ00346 protein, similar to21361124 four and a half LIM domains 2; down-regulated in rhabdomyosarcoma LIM protein4504269 H2B histone family, member J123678 heat shock 90kD protein HSP 90-R (HSP 86)337748 human serum amyloid A21740032 hypothetical protein13376747 hypothetical protein FLJ12660 [Homo sapiens] >gi|10434286|dbj|BAB14203.1| unnamed protein product [Homo sapiens]14602715 hypothetical protein FLJ1294912654391 hypothetical protein FLJ20450, similar to21411003 hypothetical protein FLJ2050916878298 hypothetical protein FLJ23469, similar to21389433 hypothetical protein FLJ3238927666102 hypothetical protein MGC4767 [Rattus norvegicus] Similar to17451530 hypothetical protein XP_07004918543694 hypothetical protein XP_1050898918518 immunoglobulin γ heavy chain [Homo sapiens]5679520 immunoglobulin heavy chain variable region33700 immunoglobulin λ light chain [Homo sapiens]27481124 KIAA1078 protein Similar to27484006 leucine-zipper protein FKSG13 [Homo sapiens] similar to7661686 Mitochondrial 39S ribosomal protein L56 (MRP-L56) (Serine â)20986529 mitogen-activated protein kinase 1; extracellular signal-regulated kinase 2; protein tyrosine kinase ERK2;

mitogen-activated protein kinase 24587083 Multidrug resistance-associated protein 521536288 muscle creatine kinase; creatine kinase M chain4505315 myomesin 2; titin-associated protein, 165 kD; myomesin (M-protein) 2 (165 kD)12667788 myosin, heavy polypeptide 9, nonmuscle187385 myristoylated alanine-rich C-kinase substrate



the temporal variations in expression, i.e., whether a givenprotein is being expressed at all under a given set of conditions.Because of this complexity, it is likely that several methods willbe required to reveal the entire complement of proteinscontained within a proteome.

As mentioned previously, the mitochondrial proteome isthought to contain an unusually high number of small, basic,and hydrophobic proteins. This is primarily due to the require-ments of the protein import machinery and the fact that theenergy generating inner membrane is rich in integral mem-brane proteins. In our analysis of the mitochondrial proteomeusing MDLC/MS/MS, we identified a substantial number ofproteins with calculated molecular weight less than 20 kDa(27% and 18% of the identifications in Expt 1 and Expt 2,respectively) and proteins with a calculated pI value greaterthan 9 (33% and 20% of the Expt 1 and Expt 2 identifications,respectively). These results are similar to those obtained by1DE/LC/MS/MS where 25% and 32% of the identified proteinswere small and basic, respectively. In fact, the distributions oftheoretical molecular weight and pI values for all proteinsidentified by both MDLC/MS/MS experiments (see Supple-mentary Figures 2 and 3) closely match the analogous distribu-tions charted for the 1DE/LC/MS/MS study and support theconclusion that the character of the mitochondrial proteomeis skewed toward small basic proteins. The close correspon-dence between the protein molecular weight distributions forthe gel and nongel based methods is particularly noteworthygiven that on average only 11% of proteins in these distributionshave molecular weights greater than 80 kDa. This resultsupports the conclusion that the mitochondrial proteome

simply does not contain many high molecular weight proteins(at least at an expression level detectable by LC/MS/MS). Hada greater proportion of high molecular weight proteins beenpresent, it is very unlikely that the non gel-based studies wouldhave failed to identify them: larger proteins tend to generatea greater number of tryptic peptides than lower molecularweight proteins, thus increasing the chances that at least oneof these peptide markers would have been selected for MS/MS during data dependent acquisition.

The mitochondrial inner membrane is composed of 50%integral and 25% peripheral membrane protein.19 Thus, themitochondrial proteome would be expected to contain anabundance of hydrophobic proteins. The most hydrophobicof these include the proteins encoded by the mitochondrialDNA: The ancient prokaryote that apparently evolved into themitochondrion must have contained a full complement ofgenetic material, most of which was eventually transferred tothe host genome. However, genes encoding 13 proteins (allcomponents of electron transport chain complexes) remained,presumably because the extremely hydrophobic character ofthese proteins would prevent their import back into theorganelle after translation. A total of 11 out of 13 mitochon-drially encoded proteins were identified by MDLC/MS/MS. Ofthe 2 unidentified subunits, Complex I ND4L contains only onetryptic cleavage site, producing only one peptide (of 23 aminoacids) potentially suitable for MS/MS. The Complex IV subunitI was the other unidentified subunit but could be expected toproduce only up to 4 peptides between 10 and 25 amino acidsin length. Subunit c of Complex V was one extremely hydro-phobic protein identified by the current study in the insoluble

Table 1 (Continued)

genbank ID annotation

2245566 NADH dehydrogenase III [Homo sapiens]228797 neutrophil granule peptide HP14505399 NIPSNAP homologue 1; 4-nitrophenylphosphatase domain and nonneuronal SNAP25-like 1998943 orosomucoid 1 precursor; Orosomucoid-1 (R-1-acid glycoprotein-1); R-1-acid glycoprotein 125376787 ovarian carcinoma immunoreactive antigen4507173 paraplegin [Homo sapiens] >gi|3273089|emb|CAA76314.1| paraplegin [Homo sapiens]17450333 Peptidyl-prolyl cis-trans isomerase A (PPIase) (Rotamase) (Cyclophilin A) (Cyclosporin A-binding protein),

similar to488425 peripheral benzodiazepine receptor - human >gi|488425|gb|AAA18228.1| peripheral benzodiazepine receptor18088748 putative receptor protein19923736 pyruvate dehydrogenase kinase, isoenzyme 236038 rho GDP dissociation inhibitor (GDI)7428728 S100 calcium binding protein A117475302 similar to fatty acid binding protein 3; Fatty acid-binding protein 3, muscle; H-FABP; mammary-derived growth inhibitor2146974 skeletal muscle LIM-protein 1 - human15072538 SLAM, signaling lymphocytic activation molecule30315658 spectrin, â, nonerythrocytic 1 isoform 230147527 succinate dehydrogenase flavoprotein subunit, mitochondrial precursor (Fp) (Flavoprotein subunit of complex II)17066105 titin6690160 TRAF6-binding protein T6BP4557871 transferrin587432 troponin T15290517 truncated cardiac troponin T135397 tubulin R-1 chain, brain-specific5174735 tubulin, â, 29507245 tyrosine 3-monooxgenase/tryptophan 5-monooxgenase activation protein, γ polypeptide; 14-3-3 protein3850565 ubiquinol-cytochrome c reductase (6.4kD) subunit XI5174745 ubiquinol-cytochrome c reductase hinge protein29841328 UDP-N-acteylglucosamine pyrophosphorylase 1 in Homo sapiens [Schistosoma japonicum] Similar to10441936 unknown16877108 unknown (protein for MGC:24572)14250650 unknown (protein for MGC:3704)22902184 unknown (protein for MGC:40410)28193244 unnamed protein product22761477 unnamed protein product23200008 Williams Beuren syndrome chromosome region 21 isoform 1



fraction of Expt 2. The MS/MS spectrum for the observedpeptide sequence is shown in Figure 4. To our knowledge thissubunit has not been identified in any previous gel-based studyof mitochondrial proteomes, including an analysis of SDS-PAGEbands from isolated intact Complex V prepared by immuno-capture.30

The average hydropathy values of the proteins identified byboth MDLC/MS/MS experiments were calculated using meanKyte-Doolittle amino acid values, and are plotted in Figure 5along with the values calculated for the 573 proteins identified

by 1DE/LC/MS/MS. The three distributions, representing twodifferent protein extraction conditions (sucrose density gradientfractionation versus differential solubilization) and two differentanalytical approaches (gel versus nongel), are nonethelessnearly identical.

One class of proteins that was not well represented in thecurrent study was proteins of low abundance. Proteins knownto be expressed at high abundance (i.e., from 2DE experiments)such as components of the oxidative phosphorylation machin-ery (OXPHOS), the adenine nucleotide translocators, and theisoforms of the voltage dependent anion channels were easilyidentified in the current study and are discussed in thesubsequent section. However, the number of identified mito-chondrial ribosomal proteins and components of the TIM(translocation of the inner membrane) and TOM (translocationof the outer membrane) complexes, expressed at much lowerlevels, are substantially reduced. A total of 35 mitochondrialribosomal proteins and 8 components of TIM and TOMcomplexes were previously identified in the mitochondrialproteome by 1DE/LC/MS/MS. In the current studies, 2 ribo-somal proteins and 1 TIM component were identified by Expt1, while Expt 2 identified only 6 ribosomal proteins and no TIMor TOM components. We were particularly surprised at the lackof identified proteins by the first experiment given that thiswas a portion of the same exact sample analyzed by 1DE/LC/MS/MS. For example, 1 ribosomal protein was identified byExpt 1 in fraction SG3, yet this fraction was known to containat least 9 mitochondrial ribosomal proteins from our previousstudy. Fraction SG3 was also of particular interest because ithas been shown to be enriched in Complex I and other proteins

Figure 4. MS/MS spectrum acquired from MDLC/MS/MS Expt 2 and correlated with (CNBr derived, M ) homoserine) peptide sequenceVAFLILFAM from mitochondrial Complex V subunit c.

Figure 5. Distributions of average hydropathy values for proteinsidentified in each of three experiments performed, calculatedusing mean Kyte-Doolittle amino acid values. MDLC/MS/MSExpt 1 and MDLC/MS/MS Expt 2 represent proteins identified inthe current work. Values from the 1DE/LC/MS/MS experimentwere reported previously13 and are included for comparison.



that may functionally interact with Complex I. Thus, anadditional analysis of fraction SG3 was carried out with one ormore adjusted variables: four times more sample was loadedthan in previous runs (3.0 µg versus 0.75 µg) to improve peptidedetection, an exclusion list based on precursor ions alreadyselected for MS/MS was used to increase the chances for newMS/MS events and a narrow survey scan mass window wasused in attempt to trigger MS/MS events on lower abundancepeptides. Yet only the single ribosomal protein was identified.It is important to note that this was not an intrinsic bias ofMDLC/MS/MS toward the particular class of ribosomal pro-teins per se, as other researchers have successfully identifiednearly the entire mitochondrial ribosome by LC/MS/MS meth-ods, using a sample of purified mitochondrial ribosomes.31 Itmay, however, represent a limitation of the MDLC/MS/MSexperimental design as applied to this sample with respect todynamic range of the analysis.

Total Coverage of the Mitochondrial Proteome. Takentogether, the gel- and non gel-based analysis of the humanheart mitochondrial proteome have revealed 680 proteins likelyto be present in or closely associated with the mitochondria.The functional classifications of the proteins identified in Expt

1 and Expt 2 are shown in Figure 6a and 6b. There are somenotable differences between these distributions, although wecannot discount the fact that this may be due to a number offactors including the different LC and MS methods used.Sample origin may also play a role. Although each sample waspooled from more than one source, pooling only 2-3 hearts isnot enough to completely eliminate individual biologicalvariation. The specific reason(s) for such different distributionsof identified proteins in these samples (both of which repre-sented a “healthy” human heart mitochondrial proteome)remains an interesting question. Given that our initial goal inthese analyses was to increase our coverage of the proteome,the distribution of identified proteins from Expt 2, which wasquite different from both the 1DE/LC/MS/MS experiment andMDLC/MS/MS Expt 1, went further toward meeting this goal.

Much better coverage of the OXPHOS machinery wasobtained in Expt 1 than in Expt 2. This may be a reflection ofthe initial sample preparation and protein fractionation meth-ods. The sucrose density gradient fractionation of proteincomplexes applied to the sample analyzed in Expt 1 wasspecifically optimized for segregating Complexes I-V. In ad-dition, the pie graph depicting all functions of identified

Figure 6. Distributions of functional classifications for proteins identified by (A) MDLC/MS/MS Expt 1 and (B) MDLC/MS/MS Expt 2.



proteins from Expt 1 is proportionally quite similar to thepreviously published graph from the 1DE/LC/MS/MS experi-ment, where the same sucrose gradient density fractionationwas used.

Coverage of the oxidative phosphorylation machinery(OXPHOS) is of particular interest because, to date, all of theOXPHOS complexes have been implicated in one or moredisease states. These include Parkinson’s disease (Complex I),4

Huntington’s disease and Friedreich’s ataxia (Complexes II andIII),32 diabetes mellitus (Complexes I and IV),5 schizophrenia(Complexes I, III and IV),33 and Alzheimer’s disease (ComplexIV and V).2,34,35 This is perhaps not surprising: dysfunction ineven a single subunit (for example, caused by genetic mutationor oxidative damage) of an OXPHOS complex could easilyrender the entire complex dysfunctional. This in turn wouldimpair the entire OXPHOS machinery. Cells such as the brain,heart, and liver that rely heavily on the important energyproducing and ROS mediating capacities of the mitochondriabecome unable to function and extremely susceptible todamage. We have found at least one marker by non gel-basedmethods for 6 of the 9 OXPHOS subunits not found in ourprevious studies12,13 for a total of 97% of OXPHOS subunits inall three studies combined. We plan to use these results as abasis for differential expression studies of these subunits invarious disease states.

Another feature of note in Figure 6 is the fairly largeproportion of cytoskeletal proteins identified by Expt 2 (14%of identified proteins). This observation may also be related tosample preparation. The sucrose gradient density fractionationwas observed to minimize cytoskeletal components in thesample by confining them to the pellet.13 This extra stage ofsample “clean up” was not carried out for the online MDLC/MS/MS study. The practical consequence of this samplepreparation was that only 1/11th of the fractions analyzed byMDLC/MS/MS in the offline study contained significantlyabundant peptides from structural proteins for MS/MS analysis.In contrast, each fraction analyzed by the online MDLC/MS/MS study contained a detectable level of peptides from theseproteins, thereby increasing the chances that each of thesepeptides would be selected for MS/MS. The presence ofcytoskeletal components is not surprising even from such ahighly purified mitochondrial preparation because of the closeassociation of the mitochondria with the cytoskeleton.1 Thishighlights the importance of using a mitochondrial preparationseparated as much as possible from these other components.Because many cytoskeletal proteins are large (∼100 kDa) andgenerate many tryptic peptides, their presence will tend toreduce the number of true mitochondrial proteins observed.The presence of a greater number of tryptic peptides for theselarge cytoskeletal components also improves the chance thatone of these marker peptides will be selected for MS/MS ratherthan a tryptic peptide from a small protein, which tends toproduce fewer total peptides. The low abundance componentswill also be that much more difficult to detect, the morenonmitochondrial proteins there are present in the mixture.

Conclusions

The current analysis of the mitochondrial proteome usingnon gel-based methods has provided additional support for 366identified mitochondrial proteins from our previous studies,and yielded 107 identifications not obtained previously.12,13 Inall three experiments combined i.e., 1DE/LC/MS/MS,12,13 and

MDLC/MS/MS Expt 1 and Expt 2, we have now mapped 680proteins associated with human heart mitochondria. Amongthese three experiments, the sucrose density gradient proteinfractionation combined with SDS-PAGE separation and LC/MS/MS (1DE/LC/MS/MS) provided the greatest proteomecoverage in terms of total number of proteins identified,dynamic range, and functional classification. Both MDLC/MS/MS experiments identified approximately the same number ofproteins with roughly a 50% overlap in terms of the specificprotein IDs. This could be a reflection of the different sampleorigins or of the different LC, MS, and data analysis methodsused in each of these experiments.

Because human mitochondria have been estimated tocontain more than 1000 proteins, the next issue to address ishow to further expand coverage of the proteome. A related issueis the accuracy of this estimate: our list of 680 proteins wouldrepresent a much greater proteome coverage if the number ofmitochondrial proteins were accurately known to be 800 ratherthan 1500 components, for example. Regardless of the actualnumber, several known mitochondrial proteins are noticeablyabsent from our list. Functional classes such as cell death/defense and DNA repair in particular appear to be underrep-resented. It is possible that many of these protein componentswere simply not being expressed in the temporal “slice” of theproteome we examined. Thus, comparative proteomics willplay a key role in elucidating these protein identifications. Anexamination of the mitochondrial proteome under conditionsof oxidative stress, for example, would be expected to up-regulate proteins in this latter class. An equally likely possibilityis that many more proteins were present but outside thedetection limits of both the gel and non gel methodologies asapplied to this sample. This is almost certainly true for factorsassociated with apoptosis. Improved protein and peptideseparation strategies such as co-immunoprecipitation or isola-tion of discrete protein complexes (rather than the relativelylow resolution sucrose density gradient separation) and highresolution gradient strong cation exchange separation ofcomplex peptide mixtures (rather than using discrete salt steps)would likely increase the dynamic range of mass spectrometricdetection. Along with higher resolution separation methods,however, comes the requirement for increased analysis time:The 1DE/LC/MS/MS method, highly resolved at the proteinlevel, identified twice as many proteins as the less resolvedMDLC/MS/MS Expt 2 analysis yet required an order ofmagnitude more analysis time. To reap the benefits of in-creased proteome coverage yet counteract the greatly reducedreturn on investment of time, new methods to improve dataacquisition must be developed to target peptides derived fromproteins that have not yet been identified.

Acknowledgment. We thank Lara Hays and Dr. GaryGlenn for technical assistance and advice. This work wassupported in part by STTR Grant ARMY03-T15 awarded by theU.S. Army Research Office to MitoKor Inc.

Supporting Information Available: Distributions ofpI and molecular weight values for identified proteins. Tablesof human heart mitochondrial proteins identified in this studyby MDLC/MS/MS. This material is available free of charge viathe Internet at http://pubs.acs.org.



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