Expanded Coverage of the Human Heart Mitochondrial Proteome

Thus, a manual cartridge-based separation strategy appeared to be a satisfactory and convenient method of fractionating the human heart mitochondrial ...
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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 degenerative disorders such as Alzheimer’s disease and Parkinson’s disease. Thus, there has been increasing interest in cataloging mitochondrial proteomes to identify potential diagnostic and therapeutic targets. We have previously reported results of a one-dimensional electrophoresis/liquid chromatography MS/MS study to 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 chromatography MS/MS was investigated as an alternative means for characterizing the same sample. Keywords: mitochondria • LC/MS/MS • MudPIT • MDLC

Mitochondria are the powerhouses of cells and also play an integral role in ion homeostasis, fatty acid oxidation, intracellular signaling and in the regulation of oxidative stress and cell death processes.1 Furthermore, recent evidence suggests that mitochondria are closely linked to the aging process and to many degenerative disorders such as Alzheimer’s disease,2 Parkinson’s disease3,4 and diabetes mellitus.5 The emerging role of mitochondrial dysfunction in disease has led to a surge of interest in studying mitochondrial proteomes6-17 to identify potential diagnostic and therapeutic targets. The exact number of mitochondrial proteins is not known, but is estimated to be on the order of 1000 proteins.18 Only 13 of these proteins are encoded by the mtDNA; the remainder is encoded by the nuclear genome and must be imported into the mitochondria.19 Temporal variation in protein expression (i.e., during respiration, biogenesis, apoptosis, etc) and tissue distribution and localization to mitochondrial sub-compartments (mitochondrial inner membrane, matrix, intermembrane space, etc) increases the complexity of the analysis. The method of choice for proteomic studies to date has been based primarily on two-dimensional electrophoresis (2DE) and either 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 recalcitrant to this type of analysis.11 A large percentage of these proteins are integral or peripheral membrane proteins because many of this organelle’s functions occur within its inner and outer membranes. Such proteins are quite difficult to solubilize and resolve well by 2DE due to their extremely hydrophobic character. In addition, many mitochondrial proteins are small (MW < 15 kD) and basic (pI > 9).20 To improve the coverage of these protein classes, a series of gels may be run with varying pH gradients for the isoelectric focusing step. Previous studies on human mitochondrial proteomes with the sample analyzed on a single 2DE gel resolved up to ca. 1500 spots of which 5060 unique proteins were identified.6,9 Improvements in protein coverage were obtained on the mitochondrial proteomes of human placenta14 and rat liver7,10 by running a series of gels obtained from aliquots of the sample enriched for various protein classes (∼100 gene products identified),7 or from the same sample by varying the first dimension pH gradient (∼130 and ∼200 gene products identified).10,14 However, despite the improvements in the total number of proteins identified, the low number of basic proteins and membrane proteins identified remained a methodological issue.

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

Two studies of mitochondrial proteomes have reported the use of alternate separation strategies. Pflieger et al.11 identified 179 gene products from yeast mitochondria using onedimensional electrophoresis/liquid chromatography MS/MS (1DE/LC/MS/MS). Their results are comparable in number with the six 2DE gels run by Fountoulakis et al.10 but encompass a much greater percentage of small, basic, and membrane proteins. Spahr et al.8 bypassed gels altogether to study the proteins released from mouse liver mitochondria undergoing atractyloside-induced membrane permeabilization. They iden-

Introduction

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 2004 American Chemical Society

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research articles tified 108 gene products after only nine LC/MS/MS experiments, and the experimental design was conducive to highthroughput studies. Three recent large scale studies on nonhuman mitochondrial proteomes, yeast,15 mouse,16 and plant17 have generated lists of 750, 591, and 416 mitochondrial proteins for these organisms, respectively. The strategy for these studies included pooling data from multiple types of experiments or performing experiments on different tissues to expand upon the lists of proteins that could be generated from any one of these experiments alone. The long term objective of our current studies is to comprehensively catalog the mitochondrial proteome from human heart. Given the improved proteome coverage using 1DE/LC/ MS/MS and nongel based approaches, we applied these techniques to a highly purified mitochondria preparation. Recently, the results of our analysis using 1DE/LC/MS/MS were presented.13 In this report, we describe the use of multidimensional liquid chromatography coupled with tandem mass spectrometry (MDLC/MS/MS) toward the mapping of the mitochondrial proteome.

Experimental Section Materials. Mitochondria were obtained from human heart (Analytical Biological Services, Wilmington, DE) and further purified using a Percoll/metrizamide gradient as described elsewhere.9 Samples for each of the two MDLC experiments described below (Expt 1 and Expt 2) were from separate pools of donors: Sample for Expt 1 was isolated from the same pool of three donor hearts as described previously,13 and sample for Expt 2 was isolated from a different pool of two hearts. The latter donors were both approximately 45 years of age, and the cause of death was unrelated to cardiovascular disease. All chemical reagents used were of analytical grade or better. MDLC/MS/MS Analysis 1 (Expt 1). In these experiments, a portion of the protein fractions generated for the 1DE/LC/MS/ MS analysis13 was used. The fractions had been obtained by solubilizing purified human heart mitochondria in n-dodecylβ-D-maltoside buffer and separating the proteins, including intact complexes, using sucrose density centrifugation.12,20 Ten sucrose gradient fractions (labeled SG1 through SG10) and one insoluble pellet (labeled LMP, for lauryl maltoside pellet) were used for Expt 1. Typically, 100 µg of each sample was dissolved in 100 µL of 50 mM Tris/0.1% SDS, pH 8.5. A 2 µL portion of 50 mM TCEP solution was added, and the sample was boiled for 10 min. The sample was then incubated in the dark at RT for 1 h after adding 5 µL of 100 mM iodoacetamide. Trypsin was added (100 µL of a 0.1 µg/µL solution in water), and the sample was incubated at 37 °C overnight. A portion of each digest (equivalent to 50 µg protein) was diluted 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, Applied Biosystems, Foster City, CA) fitted with a syringe adapter. After loading, peptides in fractions SG1-SG10 were eluted with successive salt steps of 100 µL (0.5 column volumes) each of 35, 52.5, 70, 87.5, 105, 122.5, 140, 157.5, 175, and 350 (4×) mM KCl in 10 mM KH2PO4 buffer (pH < 3) containing 25% aqueous acetonitrile. Fractions were collected in 100 µL increments, and peptides were contained in fractions 3-12 (corresponding to the solvent fronts of each of the 10 salt steps). An alternate elution series was initially tried using LMP and SG10, where successive salt steps of 500 µL each (2.5 column volumes) of 52.5, 87.5, 140, 175, and 350 (2×) mM KCl were used to elute 496

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the peptides, and fractions were collected in 250 µL increments. However, this method introduced too much salt into the fractions, resulting in relatively poor binding to the C18 precolumn during LC/MS/MS and prompted the switch to the improved elution series for SG1-SG10. Data collected on SG10 with both methods was included in the final analysis. Sample LMP was not refractionated using the final elution series due to sample constraints. In addition to fractionating the eleven samples (SG1-SG10, LMP) as described above, a 50 µg portion of SG3 and of LMP were treated by using the SCX cartridge only as a clean up step to 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 0.32.0 µg total) were loaded at 20 µL/min with 0.05% formic acid onto 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 backflushed onto a 75 µm × 15 cm nanocolumn - 3 µm × 100 Å C18 (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% aqueous acetonitrile + 0.05% formic acid, solvent B ) 98% aqueous acetonitrile + 0.05% formic acid). Spectra were acquired on an Applied Biosystems/MDS QStar. Information Dependent Acquisition (IDA) was performed using the following parameters: 1 s MS; 3 s MS/MS on 2+ or 3+ ions at 400-1200 m/z, 5 cps minimum intensity, 60-120 s dynamic exclusion. All MS/ MS spectra recorded for these samples were used in the subsequent data analysis. Additional IDA experiments were performed on the unfractionated portions of SG3 and LMP. LMP (0.75-1.0 µg) was loaded and eluted as described above; IDA was carried out as described above but varying the m/z range used for precursor ion selection. The following ranges were used: m/z 400-1200, 350-460, 450-560, 550-660, 650-760, 750-860, 850-1200. For SG3, LC/MS/MS was performed (as for the SCX separated fractions) on a 0.75 µg and a 3.0 µg portion of the unfractionated sample. These two experiments were then repeated using a precursor ion exclusion list based on all species selected for MS/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 sample using two narrow m/z ranges for precursor ion selection, m/z 550-650 and 650-750. All data acquired was included in the final analysis. Expt 1 Data Interpretation. Sonar MS/MS21 (Genomic Solutions, Ann Arbor, MI) was used to search the spectra against 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 a protein expectation value e1 and g10-3, and a peptide expectation value e1 were manually inspected.22 The resulting list of 253 proteins obtained from this analysis was less than half that found previously by 1DE/LC/MS/MS. It was also observed that several proteins known to be present but likely to be supported by only one or two peptide sequences were absent from the list. Because protein expression values calculated by Sonar depend on the total number of spectra submit-

Human Heart Mitochondrial Proteome

ted for a search21 we surmised that proteins identified by only a single spectrum in such a large number of submitted spectra would not have a “significant” expectation value and would go unreported. The same data set was therefore submitted to Mascot MS/ MS Ions Search23 (Matrix Science, London) using the human subset of the NCBInr protein database and search parameters analogous to those listed above. Peptide matches with a score of 33 or greater (95% confidence) and corresponding to protein identifications not found using Sonar MSMS were manually inspected and if accepted, merged with the list of peptides identified by Sonar. MDLC/MS/MS Analysis 2 (Expt 2). This mode of MDLC was essentially the same as described by Washburn et al.24 with slight modification. Mixed bed columns were constructed by sequentially packing a 75 µm internal diameter PicoFrit (New Objective, Woburn, MA) with 10-15 cm of C18 (Michrom Magic) followed by 3-5 cm of SCX resin (PolySULFOETHYL Aspartamide, PolyLC, Columbia, MD) by means of a helium pressure cell (Brechbuehler, Spring, TX). Metrizamide-purified mitochondria were solubilized in 8 M urea; the resulting supernatant was labeled the “soluble fraction” and the remaining material was labeled the “insoluble fraction.” The insoluble fraction was subject to CNBr digestion in 90% formic acid. After an overnight incubation at room temperature in the dark, the solution was adjusted to pH 8.5 by addition of solid ammonium bicarbonate (NH4HCO3) taking care to avoid losses during frothing. After dilution of the soluble fraction to 2 M urea with 0.1 M NH4HCO3 and dilution of the insoluble fraction with a similar quantity of MilliQ water, the samples were treated identically. DTT was added to a final concentration of 1 mM and incubated for 1 h at 54 °C after which iodoacetamide was added to a final concentration of 10 mM and incubated for 30 min in the dark. Endoprotease Lys C (Wako) was added as 0.23 U of activity to 1 mg protein, and the resulting mixture was incubated at 37 °C (pH 8.5) for 24 h. (Protein content was colorimetrically assayed using Biorad DC, detergent compatible reagent, in the initial soluble and insoluble fractions which were 1.66 mg and 1.87 mg, respectively.) The mitochondrial proteins from each preparation were then aliquoted into 1.5 mL Eppendorf 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. After centrifugation, the beads were discarded and the digests cleaned up and concentrated using C18 solid-phase extraction cartridges (SPEC-Plus PT C18, Anysys Diagnostics, Lake Forest, CA) per the manufacturer’s instructions. Finally, the digests were concentrated from 200 µL to less than 10 µL using a speedvac, 50 µL of solvent A (95% water, 5% acetonitrile, 0.2% formic acid) was added, and the digests were stored at -80 °C until use. For MDLC/MS/MS analysis, digests were loaded directly onto the mixed bed Picofrit column previously equilibrated with solvent A by means of a helium pressure cell at 1000 PSI that corresponded to a flow rate of approximately 300 nL/min. Approximately 200 µg total peptide digest in 5-10 µL volume was loaded, an overestimate corresponding to the initial protein determination assuming total recovery from all steps. After the samples were loaded, the PicoFrit column was inserted into a simple nanospray source based on that described in the literature25 (constructed by the University of California, San Diego Department of Chemistry and Biochemistry fabrication facility), and connected to the liquid junction and capillary LC via an Upchurch Cross fitting, which also

research articles serves as a flow splitter. The Micro-Tech Ultra Plus II Proteomic System (Vista, CA) consists of 2 binary pumping systems configured 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 rate was adjusted so that after splitting it was typically 400-700 nL/ min (20-25 µL per minute presplit). The gradients were similar to the fully automated chromatography runs described in the literature.24,25 The first step of 117 min consisted of an 80 min gradient from 1 to 80% buffer B, followed by 10 min at 80% and then reequilibration with solvent A. The next steps were 124 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% buffer B which was held at 60% for 10 min followed by reequilibration with 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 fraction some spraying problems were experienced, and so these experiments were accelerated by using only 75% and 100% steps after the 50% step. For the final salt step, the reservoir containing buffer C was replaced with 1 M ammonium acetate/ 5% acetonitrile/0.2% formic acid and the column was typically washed for 15 min to remove strongly bound peptides from the SCX resin. The 90 min gradient (0 to 60% B) was then run as above. Finally, a 30 min wash with solvent A followed by a gradient from 1 to 80% buffer B over 80 min, held at 80% B for 10 min, then at 100% B for 20 min, was used to elute the remaining peptides from the column. Mass spectra were acquired on a Finnigan LCQ DECA ion trap mass spectrometer in the m/z 400-1400 range. After one full scan MS of the column effluent was recorded, three MS/MS spectra of the third most, second most and most intense MS peaks were sequentially recorded over the m/z 100-2000 range with an isolation width of 2.7 and normalized collision energy setting of 35. Dynamic exclusion was employed to select the maximum number of unique peptide peaks from the chromatograms. After replicate MS/MS spectra were acquired for a precursor ion, the m/z value of the ion was placed on an exclusion list for 7 min. Expt 2 Data Interpretation. Each chromatogram was subsequently analyzed with the program SEQUEST26 using the human subset of the NCBInr protein database (21 May, 2003) that had been “indexed for speed” with carbamidomethylation as a static modification of cysteine (+57.0 Da) using TurboSEQUEST software in Bioworks 3.1(ThermoFinnigan). For the soluble fraction indexing also specified oxidation of methionine (+16 Da) and carbamylation of lysine (+43 Da, included because urea was used for solubilizing) as differential modifications. For the insoluble fraction conversion of methionine to homoserine in the presence of cyanogen bromide was specified as a static modification (-30 Da) and unmodified methionine (+30 Da) and oxidation of tryptophan (+16 Da) were specified as differential modifications with the enzyme editor adjusted to specify methionine as well as arginine and lysine as cleavage sites. A set of web-based batch programs, written in Perl, were designed to enable automated raw data preparation, searching and reporting of SEQUEST runs. All peptide matches were filtered based on their SEQUEST cross-correlation (Xcorr) values and ∆corr values (a measure of the difference in Xcorr between the best and next best peptide match). Peptide matches with ∆corr values g0.1 and Xcorr values g1.7 (Charge Journal of Proteome Research • Vol. 3, No. 3, 2004 497

research articles State 1) or g2.0 (Charge State 2) or g3.0 (Charge State 3) were tentatively accepted. Spectral data from each of the LC/MS/MS runs were also searched against the same database (NCBInr, human subset, May 2003 release) using the SonarMSMS algorithm running on Linux. Spectral data were first merged into a set of input files, and thus processed in batch mode, using a web-client emulation program written in Perl. Peptide matches with a Peptide Expect value of e0.01 and Protein Expect value of gi|1244508|gb|AAA93254.1| assembly protein 50 clathrin heavy chain; clathrin, heavy polypeptide-like 2 clathrin, heavy polypeptide-like 1 isoform b Collagen, type VI, R-1 precursor COX17 homolog, cytochrome c oxidase assembly protein; human homologue of yeast mitochondrial copper recruitment gene cysteine desulfurase; putative tRNA splicing protein cytochrome c oxidase subunit Va cytochrome c oxidase subunit VIa polypeptide 2 cytochrome-c oxidase (EC 1.9.3.1) chain III - human mitochondrion D-lactate dehydrogenase [Homo sapiens] >gi|23506788|gb|AAM50322.1| D-lactate dehydrogenase [Homo sapiens] desmoyokin - human (fragments) dihydropyrimidinase-like 2 dimerization cofactor of hepatocyte nuclear factor 1 (HNF1) from muscle dJ127D3.2 (Flavin-containing Monooxygenase family protein) [Rattus norvegicus] Similar to dynein, cytoplasmic, heavy chain 1 eIF-5A2 protein; eIF5AII elongation factor 1 R Similar to fibronectin precursor FLJ00346 protein, similar to four and a half LIM domains 2; down-regulated in rhabdomyosarcoma LIM protein H2B histone family, member J heat shock 90kD protein HSP 90-R (HSP 86) human serum amyloid A hypothetical protein hypothetical protein FLJ12660 [Homo sapiens] >gi|10434286|dbj|BAB14203.1| unnamed protein product [Homo sapiens] hypothetical protein FLJ12949 hypothetical protein FLJ20450, similar to hypothetical protein FLJ20509 hypothetical protein FLJ23469, similar to hypothetical protein FLJ32389 hypothetical protein MGC4767 [Rattus norvegicus] Similar to hypothetical protein XP_070049 hypothetical protein XP_105089 immunoglobulin γ heavy chain [Homo sapiens] immunoglobulin heavy chain variable region immunoglobulin λ light chain [Homo sapiens] KIAA1078 protein Similar to leucine-zipper protein FKSG13 [Homo sapiens] similar to Mitochondrial 39S ribosomal protein L56 (MRP-L56) (Serine β) mitogen-activated protein kinase 1; extracellular signal-regulated kinase 2; protein tyrosine kinase ERK2; mitogen-activated protein kinase 2 Multidrug resistance-associated protein 5 muscle creatine kinase; creatine kinase M chain myomesin 2; titin-associated protein, 165 kD; myomesin (M-protein) 2 (165 kD) myosin, heavy polypeptide 9, nonmuscle myristoylated alanine-rich C-kinase substrate

3646132 18999392 4885149 66257 23821029 627367 30582727 14149825 27677886 27479619 9966867 30149784 31397 27478724 21361124 4504269 123678 337748 21740032 13376747 14602715 12654391 21411003 16878298 21389433 27666102 17451530 18543694 8918518 5679520 33700 27481124 27484006 7661686 20986529 4587083 21536288 4505315 12667788 187385 500

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Human Heart Mitochondrial Proteome Table 1 (Continued) genbank ID

annotation

2245566 228797 4505399 998943 25376787 4507173 17450333

NADH dehydrogenase III [Homo sapiens] neutrophil granule peptide HP1 NIPSNAP homologue 1; 4-nitrophenylphosphatase domain and nonneuronal SNAP25-like 1 orosomucoid 1 precursor; Orosomucoid-1 (R-1-acid glycoprotein-1); R-1-acid glycoprotein 1 ovarian carcinoma immunoreactive antigen paraplegin [Homo sapiens] >gi|3273089|emb|CAA76314.1| paraplegin [Homo sapiens] Peptidyl-prolyl cis-trans isomerase A (PPIase) (Rotamase) (Cyclophilin A) (Cyclosporin A-binding protein), similar to peripheral benzodiazepine receptor - human >gi|488425|gb|AAA18228.1| peripheral benzodiazepine receptor putative receptor protein pyruvate dehydrogenase kinase, isoenzyme 2 rho GDP dissociation inhibitor (GDI) S100 calcium binding protein A1 similar to fatty acid binding protein 3; Fatty acid-binding protein 3, muscle; H-FABP; mammary-derived growth inhibitor skeletal muscle LIM-protein 1 - human SLAM, signaling lymphocytic activation molecule spectrin, β, nonerythrocytic 1 isoform 2 succinate dehydrogenase flavoprotein subunit, mitochondrial precursor (Fp) (Flavoprotein subunit of complex II) titin TRAF6-binding protein T6BP transferrin troponin T truncated cardiac troponin T tubulin R-1 chain, brain-specific tubulin, β, 2 tyrosine 3-monooxgenase/tryptophan 5-monooxgenase activation protein, γ polypeptide; 14-3-3 protein ubiquinol-cytochrome c reductase (6.4kD) subunit XI ubiquinol-cytochrome c reductase hinge protein UDP-N-acteylglucosamine pyrophosphorylase 1 in Homo sapiens [Schistosoma japonicum] Similar to unknown unknown (protein for MGC:24572) unknown (protein for MGC:3704) unknown (protein for MGC:40410) unnamed protein product unnamed protein product Williams Beuren syndrome chromosome region 21 isoform 1

488425 18088748 19923736 36038 7428728 17475302 2146974 15072538 30315658 30147527 17066105 6690160 4557871 587432 15290517 135397 5174735 9507245 3850565 5174745 29841328 10441936 16877108 14250650 22902184 28193244 22761477 23200008

the temporal variations in expression, i.e., whether a given protein is being expressed at all under a given set of conditions. Because of this complexity, it is likely that several methods will be required to reveal the entire complement of proteins contained within a proteome. As mentioned previously, the mitochondrial proteome is thought to contain an unusually high number of small, basic, and hydrophobic proteins. This is primarily due to the requirements of the protein import machinery and the fact that the energy generating inner membrane is rich in integral membrane proteins. In our analysis of the mitochondrial proteome using MDLC/MS/MS, we identified a substantial number of proteins 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 greater than 9 (33% and 20% of the Expt 1 and Expt 2 identifications, respectively). These results are similar to those obtained by 1DE/LC/MS/MS where 25% and 32% of the identified proteins were small and basic, respectively. In fact, the distributions of theoretical molecular weight and pI values for all proteins identified by both MDLC/MS/MS experiments (see Supplementary Figures 2 and 3) closely match the analogous distributions charted for the 1DE/LC/MS/MS study and support the conclusion that the character of the mitochondrial proteome is skewed toward small basic proteins. The close correspondence between the protein molecular weight distributions for the gel and nongel based methods is particularly noteworthy given that on average only 11% of proteins in these distributions have molecular weights greater than 80 kDa. This result supports 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). Had a greater proportion of high molecular weight proteins been present, it is very unlikely that the non gel-based studies would have failed to identify them: larger proteins tend to generate a greater number of tryptic peptides than lower molecular weight proteins, thus increasing the chances that at least one of 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, the mitochondrial proteome would be expected to contain an abundance of hydrophobic proteins. The most hydrophobic of these include the proteins encoded by the mitochondrial DNA: The ancient prokaryote that apparently evolved into the mitochondrion must have contained a full complement of genetic material, most of which was eventually transferred to the host genome. However, genes encoding 13 proteins (all components of electron transport chain complexes) remained, presumably because the extremely hydrophobic character of these proteins would prevent their import back into the organelle after translation. A total of 11 out of 13 mitochondrially encoded proteins were identified by MDLC/MS/MS. Of the 2 unidentified subunits, Complex I ND4L contains only one tryptic cleavage site, producing only one peptide (of 23 amino acids) potentially suitable for MS/MS. The Complex IV subunit I was the other unidentified subunit but could be expected to produce only up to 4 peptides between 10 and 25 amino acids in length. Subunit c of Complex V was one extremely hydrophobic protein identified by the current study in the insoluble Journal of Proteome Research • Vol. 3, No. 3, 2004 501

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

by 1DE/LC/MS/MS. The three distributions, representing two different protein extraction conditions (sucrose density gradient fractionation versus differential solubilization) and two different analytical approaches (gel versus nongel), are nonetheless nearly identical.

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

fraction of Expt 2. The MS/MS spectrum for the observed peptide sequence is shown in Figure 4. To our knowledge this subunit has not been identified in any previous gel-based study of mitochondrial proteomes, including an analysis of SDS-PAGE bands from isolated intact Complex V prepared by immunocapture.30 The average hydropathy values of the proteins identified by both MDLC/MS/MS experiments were calculated using mean Kyte-Doolittle amino acid values, and are plotted in Figure 5 along with the values calculated for the 573 proteins identified 502

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One class of proteins that was not well represented in the current study was proteins of low abundance. Proteins known to be expressed at high abundance (i.e., from 2DE experiments) such as components of the oxidative phosphorylation machinery (OXPHOS), the adenine nucleotide translocators, and the isoforms of the voltage dependent anion channels were easily identified in the current study and are discussed in the subsequent section. However, the number of identified mitochondrial ribosomal proteins and components of the TIM (translocation of the inner membrane) and TOM (translocation of the outer membrane) complexes, expressed at much lower levels, are substantially reduced. A total of 35 mitochondrial ribosomal proteins and 8 components of TIM and TOM complexes were previously identified in the mitochondrial proteome by 1DE/LC/MS/MS. In the current studies, 2 ribosomal proteins and 1 TIM component were identified by Expt 1, while Expt 2 identified only 6 ribosomal proteins and no TIM or TOM components. We were particularly surprised at the lack of identified proteins by the first experiment given that this was a portion of the same exact sample analyzed by 1DE/LC/ MS/MS. For example, 1 ribosomal protein was identified by Expt 1 in fraction SG3, yet this fraction was known to contain at least 9 mitochondrial ribosomal proteins from our previous study. Fraction SG3 was also of particular interest because it has been shown to be enriched in Complex I and other proteins

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

that may functionally interact with Complex I. Thus, an additional analysis of fraction SG3 was carried out with one or more adjusted variables: four times more sample was loaded than in previous runs (3.0 µg versus 0.75 µg) to improve peptide detection, an exclusion list based on precursor ions already selected for MS/MS was used to increase the chances for new MS/MS events and a narrow survey scan mass window was used in attempt to trigger MS/MS events on lower abundance peptides. Yet only the single ribosomal protein was identified. It is important to note that this was not an intrinsic bias of MDLC/MS/MS toward the particular class of ribosomal proteins per se, as other researchers have successfully identified nearly the entire mitochondrial ribosome by LC/MS/MS methods, using a sample of purified mitochondrial ribosomes.31 It may, however, represent a limitation of the MDLC/MS/MS experimental design as applied to this sample with respect to dynamic range of the analysis. Total Coverage of the Mitochondrial Proteome. Taken together, the gel- and non gel-based analysis of the human heart mitochondrial proteome have revealed 680 proteins likely to 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 some notable differences between these distributions, although we cannot discount the fact that this may be due to a number of factors including the different LC and MS methods used. Sample origin may also play a role. Although each sample was pooled from more than one source, pooling only 2-3 hearts is not enough to completely eliminate individual biological variation. The specific reason(s) for such different distributions of identified proteins in these samples (both of which represented a “healthy” human heart mitochondrial proteome) remains an interesting question. Given that our initial goal in these analyses was to increase our coverage of the proteome, the distribution of identified proteins from Expt 2, which was quite different from both the 1DE/LC/MS/MS experiment and MDLC/MS/MS Expt 1, went further toward meeting this goal. Much better coverage of the OXPHOS machinery was obtained in Expt 1 than in Expt 2. This may be a reflection of the initial sample preparation and protein fractionation methods. The sucrose density gradient fractionation of protein complexes applied to the sample analyzed in Expt 1 was specifically optimized for segregating Complexes I-V. In addition, the pie graph depicting all functions of identified Journal of Proteome Research • Vol. 3, No. 3, 2004 503

research articles proteins from Expt 1 is proportionally quite similar to the previously published graph from the 1DE/LC/MS/MS experiment, where the same sucrose gradient density fractionation was used. Coverage of the oxidative phosphorylation machinery (OXPHOS) is of particular interest because, to date, all of the OXPHOS complexes have been implicated in one or more disease states. These include Parkinson’s disease (Complex I),4 Huntington’s disease and Friedreich’s ataxia (Complexes II and III),32 diabetes mellitus (Complexes I and IV),5 schizophrenia (Complexes I, III and IV),33 and Alzheimer’s disease (Complex IV and V).2,34,35 This is perhaps not surprising: dysfunction in even a single subunit (for example, caused by genetic mutation or oxidative damage) of an OXPHOS complex could easily render the entire complex dysfunctional. This in turn would impair the entire OXPHOS machinery. Cells such as the brain, heart, and liver that rely heavily on the important energy producing and ROS mediating capacities of the mitochondria become unable to function and extremely susceptible to damage. We have found at least one marker by non gel-based methods for 6 of the 9 OXPHOS subunits not found in our previous studies12,13 for a total of 97% of OXPHOS subunits in all three studies combined. We plan to use these results as a basis for differential expression studies of these subunits in various disease states. Another feature of note in Figure 6 is the fairly large proportion of cytoskeletal proteins identified by Expt 2 (14% of identified proteins). This observation may also be related to sample preparation. The sucrose gradient density fractionation was observed to minimize cytoskeletal components in the sample by confining them to the pellet.13 This extra stage of sample “clean up” was not carried out for the online MDLC/ MS/MS study. The practical consequence of this sample preparation was that only 1/11th of the fractions analyzed by MDLC/MS/MS in the offline study contained significantly abundant 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 these proteins, thereby increasing the chances that each of these peptides would be selected for MS/MS. The presence of cytoskeletal components is not surprising even from such a highly purified mitochondrial preparation because of the close association of the mitochondria with the cytoskeleton.1 This highlights the importance of using a mitochondrial preparation separated as much as possible from these other components. Because many cytoskeletal proteins are large (∼100 kDa) and generate many tryptic peptides, their presence will tend to reduce the number of true mitochondrial proteins observed. The presence of a greater number of tryptic peptides for these large cytoskeletal components also improves the chance that one of these marker peptides will be selected for MS/MS rather than a tryptic peptide from a small protein, which tends to produce fewer total peptides. The low abundance components will also be that much more difficult to detect, the more nonmitochondrial proteins there are present in the mixture.

Gaucher et al.

MDLC/MS/MS Expt 1 and Expt 2, we have now mapped 680 proteins associated with human heart mitochondria. Among these three experiments, the sucrose density gradient protein fractionation combined with SDS-PAGE separation and LC/ MS/MS (1DE/LC/MS/MS) provided the greatest proteome coverage in terms of total number of proteins identified, dynamic range, and functional classification. Both MDLC/MS/ MS experiments identified approximately the same number of proteins with roughly a 50% overlap in terms of the specific protein IDs. This could be a reflection of the different sample origins or of the different LC, MS, and data analysis methods used in each of these experiments. Because human mitochondria have been estimated to contain more than 1000 proteins, the next issue to address is how to further expand coverage of the proteome. A related issue is the accuracy of this estimate: our list of 680 proteins would represent a much greater proteome coverage if the number of mitochondrial proteins were accurately known to be 800 rather than 1500 components, for example. Regardless of the actual number, several known mitochondrial proteins are noticeably absent from our list. Functional classes such as cell death/ defense and DNA repair in particular appear to be underrepresented. It is possible that many of these protein components were simply not being expressed in the temporal “slice” of the proteome we examined. Thus, comparative proteomics will play a key role in elucidating these protein identifications. An examination of the mitochondrial proteome under conditions of oxidative stress, for example, would be expected to upregulate proteins in this latter class. An equally likely possibility is that many more proteins were present but outside the detection limits of both the gel and non gel methodologies as applied to this sample. This is almost certainly true for factors associated with apoptosis. Improved protein and peptide separation strategies such as co-immunoprecipitation or isolation of discrete protein complexes (rather than the relatively low resolution sucrose density gradient separation) and high resolution gradient strong cation exchange separation of complex peptide mixtures (rather than using discrete salt steps) would likely increase the dynamic range of mass spectrometric detection. Along with higher resolution separation methods, however, comes the requirement for increased analysis time: The 1DE/LC/MS/MS method, highly resolved at the protein level, identified twice as many proteins as the less resolved MDLC/MS/MS Expt 2 analysis yet required an order of magnitude more analysis time. To reap the benefits of increased proteome coverage yet counteract the greatly reduced return on investment of time, new methods to improve data acquisition must be developed to target peptides derived from proteins that have not yet been identified.

Acknowledgment. We thank Lara Hays and Dr. Gary Glenn for technical assistance and advice. This work was supported in part by STTR Grant ARMY03-T15 awarded by the U.S. Army Research Office to MitoKor Inc.

Conclusions The current analysis of the mitochondrial proteome using non gel-based methods has provided additional support for 366 identified mitochondrial proteins from our previous studies, and yielded 107 identifications not obtained previously.12,13 In all three experiments combined i.e., 1DE/LC/MS/MS,12,13 and 504

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Supporting Information Available: Distributions of pI and molecular weight values for identified proteins. Tables of human heart mitochondrial proteins identified in this study by MDLC/MS/MS. This material is available free of charge via the Internet at http://pubs.acs.org.

research articles

Human Heart Mitochondrial Proteome

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