Rapid Purification and Mass Spectrometric Characterization of Mitochondrial NADH Dehydrogenase (Complex I) from Rodent Brain and a Dopaminergic Neuronal Cell Line*,S

Birgit Schilling{ddagger},§, Srinivas Bharath M.M.{ddagger},§, Richard H. Row{ddagger}, James Murray, Michael P. Cusack{ddagger}, Roderick A. Capaldi, Curt R. Freed||, Kedar N. Prasad**, Julie K. Andersen{ddagger} and Bradford W. Gibson{ddagger},{ddagger}{ddagger},§§

From the {ddagger} Buck Institute for Age Research, Novato, CA 94945; Department of Molecular Biology, University of Oregon, Eugene, OR 97403; || Division of Clinical Pharmacology, Departments of Medicine and Pharmacology, and the Neuroscience Program, University of Colorado Health Sciences Center, Denver, CO 80262; ** Center for Vitamins and Cancer Research, Department of Radiology, University of Colorado Health Sciences Center, Denver, CO 80262; {ddagger}{ddagger} Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143


    ABSTRACT
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Oxidative stress and mitochondrial dysfunction signify important biochemical events associated with the loss of dopaminergic neurons in Parkinson’s disease (PD). Studies using in vitro and in vivo PD models or tissues from diseased patients have demonstrated a selective inhibition of mitochondrial NADH dehydrogenase (Complex I of the OXPHOS electron transport chain) that affects normal mitochondrial physiology leading to neuronal death. In an earlier study, we demonstrated that oxidative stress due to glutathione depletion in dopaminergic cells, a hallmark of PD, leads to Complex I inhibition via cysteine thiol oxidation (Jha et al. (2000) J. Biol. Chem. 275, 26096–26101). Complex I is a ~980-kDa multimeric enzyme spanning the inner mitochondrial membrane comprising at least 45 protein subunits. As a prerequisite to investigating modifications to Complex I using a rodent disease model for PD, we developed two independent rapid and mild isolation procedures based on sucrose gradient fractionation and immunoprecipitation to isolate Complex I from mouse brain and a cultured rat mesencephalic dopaminergic neuronal cell line. Both protocols are capable of purifying Complex I from small amounts of rodent tissue and cell cultures. Blue Native gel electrophoresis, one-dimensional and two-dimensional SDS-PAGE were employed to assess the purity and composition of isolated Complex I followed by extensive mass spectrometric characterization. Altogether, 41 of 45 rodent Complex I subunits achieved MS/MS sequence coverage. To our knowledge, this study provides the first detailed mass spectrometric analysis of neuronal Complex I proteins and provides a means to investigate the role of cysteine oxidation and other posttranslational modifications in pathologies associated with mitochondrial dysfunction.


Parkinson’s disease (PD)1 is a complex, age-associated, neurodegenerative disorder involving a gradual loss of dopaminergic neurons of the substantia nigra (SN) (1). Typically, the SN of early PD patients maintains significantly decreased levels of the thiol antioxidant glutathione (GSH) that normally acts to detoxify H2O2 and other reactive oxygen and nitrogen species (2, 3). Increases in lipid peroxidation and oxidative damage of both nucleic acids and proteins during PD have been documented (46). It is believed that inhibition of mitochondrial NADH dehydrogenase (Complex I) activity plays a major role in PD pathology. Schapira et al. (7) have demonstrated a significant decrease (30%) in enzyme activity of Complex I from postmortem PD samples. In addition, in vivo animal models involving Complex I inhibitors such as rotenone (8) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exhibit PD like symptoms (9).

There is strong evidence that mitochondrial Complex I inhibition is induced by accumulated damage caused by age-related oxidative stress through reactive oxygen (ROS) and reactive nitrogen species (RNS) (10, 11). Jha et al. (12) recently demonstrated in a rodent PD model that mild oxidative stress to dopaminergic PC12 cells incurred by a reduction in cellular GSH levels leads to selective inhibition of mitochondrial Complex I activity. This reported Complex I inhibition is likely due to reversible oxidation of cysteine sulfhydryls within the complex. In addition, Taylor et al. (13) have observed cysteine glutathionylation of Complex I subunits (75 and 51 kDa) under conditions of oxidative stress.

In general, endogenous and exogenous oxidative stress is a major contributor to human diseases and aging (14). It is generally thought that ROS and RNS mediate protein damage by targeting a variety of amino acids within susceptible proteins (for review see Ref. 15). Cysteine residues are particularly susceptible to oxidation (16), primarily through reversible modifications (e.g. thiolation and nitrosylation), although irreversible oxidation can eventually lead to products that cannot be easily repaired in vivo, such as cysteine sulfinic (SO2H) or sulfonic acids (SO3H) (17). To better understand the role of cysteine oxidation during Complex I inhibition in PD, we and others have recently developed strategies to determine the redox status of cysteine residues in specific proteins on a molecular level using differential alkylation labeling with stable isotopes in combination with MS (15, 1821). In an alternative approach, Murphy and colleagues (22) have described an approach using a 4-iodobutyl triphenylphosphonium reagent to label thiols of mitochondrial proteins in vivo to measure their thiol redox states during oxidative stress.

Complex I is the largest of the mitochondrial electron transport chain (ETC) enzyme complexes with an approximate molecular mass of 980 kDa. This membrane bound multimeric enzyme is referred to as an NADH:ubiquinone oxidoreductase and consists of 45–46 independent protein subunits. Bovine mitochondrial Complex I has been analyzed extensively (23) and contains seven protein subunits that are encoded by mitochondrial DNA, while the remainder are encoded by nuclear DNA (24). Overall, the 45–46 protein subunits of human Complex I contain more than 120 cysteine residues. Three-dimensional structural data of mammalian Complex I have been limited to low-resolution electron cryo-microscopic analysis of bovine Complex I (25). These and other data show Complex I to consist of several subcomplexes (26) and with an overall "L"-shaped topology showing one arm embedded in the inner mitochondrial membrane and the other peripheral arm projecting into the matrix.

To better investigate Complex I dysfunction in rodent models of PD, it is essential to have a method to rapidly purify and isolate intact Complex I from rodent brain or cultured neuronal cells. Several rapid purification methods of OXPHOS ETC complexes derived from human or bovine heart have been published over the last few years. For example, Hanson et al. (27) employed a sucrose gradient fractionation centrifugation to obtain Complex I from human heart tissue, and Taylor et al. (28) used a similar sucrose gradient strategy followed by mass spectrometric analysis to characterize human ETC complexes. In a previous study, one of our laboratories described an immunocapture method for the one-step purification of Complex I from human heart tissue (29). Similar methods have also been demonstrated for the isolation of Complex V (ATP synthase) (30) and the pyruvate dehydrogenase complex (31). More recently, our groups have developed similar immunoisolation procedures in combination with mass spectrometric characterization for bovine Complex IV (cytochrome c oxidase) (Murray et al., unpublished data), and bovine and rodent Complexes II (succinate dehydrogenase) and III (ubiquinol-cytochrome c reductase) (Schilling and Gibson, unpublished data). In addition, an approach combining Blue Native gel electrophoresis with SDS-PAGE separations was recently described (32). Using these and other methods, several posttranslational modifications (PTMs) have been identified for bovine Complex I, including N-terminal acetylation and myristylation (23), tryptophan oxidation to N-formylkynurenine (33), and phosphorylation (34, 35). In addition, treatment with cAMP-dependent protein kinase (36) or peroxynitrite (37) has led to the identification of novel phosphorylation and tyrosine nitration sites, respectively, suggesting important roles for both enzymatic and nonenzymatic modifications to Complex I.

In this current study, we have investigated the subunit composition of rodent mitochondrial Complex I in mouse brain and heart, and in the rat dopaminergic neuronal cell line 1RB3AN27 (N27). To our knowledge, no detailed mass spectrometric investigations have been performed on rodent Complex I, or on neuronal Complex I of any species. This work describes the rapid and mild isolation and characterization of intact rodent Complex I from mouse heart and brain mitochondria, and from neuronal midbrain dopaminergic rat cell line N27 mitochondria using sucrose gradient fractionation and/or immunoprecipitation (29). Both isolation protocols were optimized to purify Complex I from small amounts of rodent tissue and cell culture materials, and to reduce and limit the formation of protein artifacts during experimental procedures. Subsequently, mass spectrometric techniques, both MALDI-MS peptide mass fingerprinting (PMF) and tandem mass spectrometry (nano-HPLC-MS, MS/MS) were used to identify and characterize these rodent Complex I protein subunits. This extensive proteomic analysis of rodent neuronal Complex I will hopefully provide means to better investigate disease-related oxidative damage and PTMs to this key ETC complex.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—
All tissue culture materials were procured from Life Technologies/Invitrogen (Carlsbad, CA) or Cellgro (Kansas City, MO). Protease inhibitor mixture was obtained from Roche Diagnostics (Indianapolis, IN). Anti-Complex I mouse monoclonal antibody (mAb) against {alpha} subcomplex subunit 9 (NDUFA9; 39 kDa) and NuPAGE 4–12% Bis-Tris (MES) gels were obtained from Molecular Probes/Invitrogen (Carlsbad, CA). Materials related to proteomics, such as sample buffers, one-dimensional (1D) and two-dimensional (2D) SDS-PAGE gels, and IPG strips (11 cm, pH 3–10), were obtained from Bio-Rad Laboratories (Hercules, CA). Gel stains such as Coomassie Brilliant Blue R was purchased from Sigma (St. Louis, MO), Sypro Ruby was obtained from Molecular Probes/Invitrogen, and silver nitrate for a silver-staining protocol was purchased from Fisher Scientific (Pittsburgh, PA). For proteolysis, sequencing-grade, modified trypsin (porcine) was purchased from Promega (Madison, WI). Nonionic detergent n-dodecyl-ß-D-maltoside and additional reagents for protein chemistry including iodoacetamide and DTT were obtained from Sigma. HPLC solvents such as ACN and water were obtained from Burdick & Jackson (Muskegon, MI). For immunoprecipitation (IP) experiments, protein G agarose beads and antibody crosslinking reagent, dimethylpimelimidate, were purchased from Sigma. For MALDI-MS experiments a matrix solution of {alpha}-cyano-4-hydroxycinnamic acid in ACN/methanol was purchased from Agilent Technologies (Palo Alto, CA).

Cell Line and Tissue Samples—
Bovine heart, mouse brain and heart, and rat dopaminergic 1RB3AN27 (N27) cells were used as sources to isolate mitochondria. The dopaminergic neuronal N27 cell line was derived from an immortalized clone of rat dopamine-producing neurons by transfecting fetal mescencephalon cells with the plasmid vector pSV3neo, which carries the LTa gene from SV40 virus (38). N27 neuronal cells possess all the physiological and biochemical properties of dopaminergic neurons (39) and can be used as PD model. N27 neuronal cells were grown in RPMI 1640 containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 µg/ml) and were maintained at 37 °C in a humidified atmosphere of 5% CO2/95% air. Cells were subcultured once a week via trypsin treatment.

Preparation and Extraction of Mitochondria—
Mitochondria were prepared by the method of Trounce et al. (40). Briefly, N27 cells were harvested, washed in Buffer H (5 mM HEPES, pH 7.2, 210 mM mannitol, 70 mM sucrose, 1 mM EGTA, and 0.5% BSA) and resuspended in the same buffer. The cell suspension was homogenized and centrifuged at 800 x g for 5 min at 4 °C. The mitochondria-enriched supernatant was then centrifuged at 10,000 x g for 20 min at 4 °C. The resulting mitochondrial pellet was resuspended in buffer H and stored as aliquots at –80 °C. For mouse brain and heart samples, freshly dissected tissue was washed and homogenized in ice-cold isolation buffer (320 mM sucrose, 5 mM Tes (N-[tris(hydroxymethyl)methyl]-2-aminoethanesulfonic acid), 1 mM EGTA, pH 7.2). The homogenate was centrifuged at 1,000 x g for 5 min at 4 °C, and then the supernatant was centrifuged at 8,500 x g for 10 min at 4 °C. For mouse brain, the pellet enriched in mitochondria was resuspended in isolation buffer and layered on top of 6% (w/v) Ficoll solution and centrifuged at 75,000 x g for 30 min at 4 °C to remove myelin, which forms a layer at the top. The pellet was resuspended in reconstitution buffer (250 mM sucrose, 10 mM Tes, pH 7.2) and stored as aliquots at –80 °C. Approximately 2 mg of rodent mitochondria (from either mouse heart, mouse brain, or dopaminergic rat cell line, respectively) were washed with 20 mM Tris-HCl, pH 7.5, 1 mM EDTA, and suspended in the same buffer containing protease inhibitor mixture. This suspension was solubilized by adding n-dodecyl-ß-D-maltoside to a final concentration of 1% at 5 mg/ml protein concentration and incubated for 30 min on ice. Insoluble material from this suspension was removed by centrifugation at 55,000 x g for 30 min at 4 °C in a Beckman TLA100.2 (Fullerton, CA).

Sucrose Density Gradient Fractionation and Western Blot Probing—
Sucrose gradient experiments were performed according to the methods previously described (27). Briefly, the supernatant obtained after solubilization of mitochondria (as described above) was subjected to a 10–35% sucrose step gradient consisting of 1-ml step fractions of 35, 32.5, 30, 27.5, 25, 22.5, 20, 17.5, 15, and 10% prepared in 10 mM Tris, pH 7.5, 1 mM EDTA, and 0.05% n-dodecyl-ß-D-maltoside. The mitochondrial sample was loaded onto the gradient in 5% sucrose and centrifuged at 38,000 rpm for 16.5 h at 4 °C in an Optima-XL 100K ultracentrifuge (SW40 Ti swinging bucket rotor) from Beckman. Fractions of 0.5 ml were collected from the top, frozen, and stored at –80 °C. Each fraction was concentrated to 100 µl using a microcon-100 concentrator (Millipore, Billerica, MA). An aliquot of 20 µl of each concentrated fraction (20% of fraction volume) was analyzed by 1D SDS-PAGE; subsequently, proteins were transferred from the gel onto a PVDF membrane and probed with an anti-Complex I mAb against the 39-kDa Complex I subunit NDUFA9 for 2 h at room temperature. The Western blot was washed several times and incubated for 2 h with anti-mouse secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences, Piscataway, NJ; part of GE Healthcare). The blot was washed and developed using the ECL Western blot detection kit (Amersham Biosciences).

Blue Native Gel Electrophoresis (BN-PAGE)—
Sucrose density gradient fractions that were positive for Complex I as indicated by Western analysis (39-kDa mAb) were pooled, concentrated, and subjected to BN-PAGE analysis according to the method of Schagger and Jagow (41, 42). Briefly, to 20 µl of concentrated pooled Complex I fractions (20% of total sample volume), 20 µl of BN sample buffer (1.5 M amino caproic acid, 0.05 M Bis-Tris, pH 7.0), 2.5 µl of 10% n-dodecyl-ß-D-maltoside, 2.5 µl of protease inhibitor mixture, and 4 µl of gel loading buffer (5% Serva Coomassie Brilliant Blue G-250, 1 M aminocaproic acid) were added and loaded onto a 4–15% Blue Native Tris gel (Bio-Rad Laboratories). Fifty micromolar tricine, 15 mM Bis-Tris, pH 7.0, 0.02% Coomassie Blue was used as cathode buffer and 50 mM Bis-Tris pH 7.0 as anode buffer. The gel was run at 4 °C at 5 mA for the first 12 h and then at 10 mA until the dye marker reached the bottom of the gel. The gel was stained with 0.25% Coomassie Brilliant Blue G-250 (Serva Electrophoresis, Heidelberg, Germany) for 1 h, destained in 50% methanol/10% acetic acid for 1 h, and then with 10% methanol/10% acetic acid overnight.

SDS-PAGE of Sucrose Gradient Fractions—
For 1D SDS-PAGE analysis, pooled sucrose gradient fractions (20% of total sample volume was loaded) were separated on 10–20% precast SDS-PAGE, on 4–12% XT Bis-Tris SDS-PAGE (Bio-Rad Laboratories), or on NuPAGE 4–12% Bis-Tris (MES) gel (Invitrogen), respectively. Typically, the sample to 1x loading buffer ratio was 1:1 (v:v). Gels were run in a X-Cell Sure Lock Mini Cell Apparatus (Invitrogen) with a SDS running buffer (50 mM MES, 50 mM Tris base, 0.1% SDS, 1 mM EDTA) at 100 V for 1.5–2h. For 2D SDS-PAGE, samples (two concentrated sucrose gradient fractions that were highly enriched in Complex I) were diluted to a final volume of 185 µl with Sigma TotProt denaturing reagent 3 and used to rehydrate IEF strips (11 cm, pH 3–10) (Bio-Rad Laboratories) overnight at room temperature. The strips were subjected to IEF for 35,000 Vh at maximum 8,000 V in a Bio-Rad Protean IEF cell system. Focused protein strips were immersed in equilibration buffer (50 mM Tris HCl, pH 8.3, 6 M urea, 2% SDS, and 0.01% bromphenol blue) for 15 min at room temperature and then run on 10–20% Tris-SDS-PAGE gradient gels (Bio-Rad Laboratories) in a Criterion gel apparatus (Bio-Rad Laboratories), and then fixed with 10% methanol, 7% acetic acid for 30 min. All SDS gels were fixed with 10% methanol, 7% acetic acid for 30 min, and subsequently stained with Sypro Ruby followed by destaining in 10% methanol/7% acetic acid).

Immunoreactivity Analyses—
The analytical mAbs were generated at the Monoclonal Antibody Facility (University of Oregon, Eugene, OR) and were further developed from those mAb originally described by Murray and Capaldi (29). For analytical Complex I-mAb testing against rodent tissue, 50 µg of mouse or bovine heart mitochondria were resolved along the entire length of each Tris-HCl 10–22% acrylamide gel. Electrophoresis was performed according to Laemmli (43). The gels were transferred in CAPS buffer from gels to PVDF membranes (0.45-µm pore size) according to Triepels (44). Each blot was simultaneously probed with multiple antibodies using the Mini-PROTEAN II multiscreen apparatus (Bio-Rad Laboratories). Complex I protein subunits were detected by the mAbs 20K 20E9 (ND6), 22K 2C7 (NDUFS4), 30K 3F9 (NDUFS3), 39K 20C11 (NDUFA9), S5 21A6 (NDUFS5), 17K 21C11 (NDUFB6), 17K 22B8 (NDUFB6), and 8K 17C8 (uncharacterized Complex I subunit) followed by a secondary goat anti-mouse polyclonal antibody conjugated to horseradish peroxidase then visualized by the ECL+ method (Amersham Biosciences).

Immunoisolation of Rodent Complex I—
The new "second generation" anti-Complex I immunocapture mAbs described above (i.e. mAb 20D1, 18G12, 17C8, 17G3, 20E9) were tested for efficiency across species against mitochondria isolated from bovine and mouse heart mitochondria (100 µg each). After optimization of the protocol, mAb 18G12 was selected as superior for IP of Complex I from mitochondria isolated from mouse heart and brain and rat cell line (N27), respectively. For immunoisolation, 50 µg of mAb 18G12BC2AA10 was bound to 5 µl of swollen protein G agarose beads. Beads were collected by gentle centrifugation at 3,000 rpm in a microfuge and resuspended in PBS. The antibody was crosslinked to the beads with 25 mM dimethylpimelimidate for 30 min at room temperature in 0.2 M sodium borate, pH 9.0. Crosslinking was terminated with 0.1 M ethanolamine solution, pH 8.0, for 3 h at room temperature. Antibody crosslinked beads were collected by gentle centrifugation at 3,000 rpm in a microfuge and resuspended in PBS. This conjugate was incubated overnight at 4 °C with the supernatant from 300 µg of solubilized mouse brain mitochondria. For this study, similar experiments were performed with 40 µg of solubilized mouse heart mitochondria, and with 200 µg of solubilized rat cell line N27 mitochondria (in addition, one immunoisolation of Complex I from 10 µg of solubilized bovine heart mitochondria was performed as described by Murray et al. (29)). Beads were washed six times with PBS supplemented with 0.05% n-dodecyl-ß-D-maltoside. Immunocaptured NADH dehydrogenase was eluted with 10 µl of 0.1 M glycine, pH 2.5, supplemented with 0.05% n-dodecyl-ß-D-maltoside and the supernatant collected. Then 10 µl of 2x SDS-PAGE sample buffer (4% SDS, 200 mM Tris, pH 6.8, 10% glycerol, bromphenol blue) were added to the sample that was then neutralized with 1 µl of 1 M Tris, pH 8.0. The 20-µl sample was resolved by SDS-PAGE either on a NuPAGE 4–12% Bis-Tris gel (Invitrogen) or on a Tris-HCl 10–22% acrylamide gel according to Laemmli (43); both gels were then stained with Coomassie Brilliant Blue R. One gel was destained and then restained with silver.

In-gel Tryptic Digestion of Mitochondrial Proteins—
Protein spots of interest were manually excised out of the gel and processed with an automatic in-gel digester robot, ProGest (Genomic Solutions, Ann Arbor, MI). The gel spots were destained and dehydrated with ACN. Subsequently, the proteins were reduced with 10 mM DTT at 60 °C for 30 min, alkylated with 100 mM iodoacetamide (37 °C, 45 min), and then incubated with 125–250 ng of sequencing-grade trypsin (Promega) at 37 °C for 4 h. The resulting tryptic peptides were then extracted from the gel by aqueous/10% formic acid extraction and analyzed by MS.

Mass Spectrometry—
Mass spectra of digested gel spots were obtained by MALDI-TOF MS on a Voyager DESTR plus instrument (Applied Biosystems, Framingham, MA). All mass spectra were acquired in positive-ionization mode with reflectron optics. The instrument was equipped with a 337-nm nitrogen laser and operated under delayed extraction conditions; delay time 190 ns, grid voltage 66–70% of full acceleration voltage (20–25 kV). All peptide samples were prepared using a matrix solution consisting of 33 mM {alpha}-cyano-4-hydroxycinnamic acid in ACN/methanol (1/1; v/v); 1 µl of analyte (0.1–1 pmol of material) was mixed with 1 µl of matrix solution, and then air-dried at room temperature on a stainless steel target. Typically, 50–100 laser shots were used to record each spectrum. The obtained mass spectra were externally calibrated with an equimolar mixture of angiotensin I, ACTH 1–17, ACTH 18–39, and ACTH 7–38.

All proteolytic peptide extracts were analyzed by reverse-phase nano-HPLC-MS/MS. Briefly, peptides were separated on an Ultimate nanocapillary HPLC system equipped with a PepMapTM C18 nano-column (75 µm inner diameter x 15 cm) (Dionex, Sunnyvale, CA) and CapTrap Micro guard column (0.5-µl bed volume; Michrom, Auburn, CA). Peptide mixtures were loaded onto the guard column and washed with the loading solvent (H2O/0.05% formic acid, 20 µl/min) for 5 min, then transferred onto the analytical C18-nanocapillary HPLC column and eluted at a flow rate of 300 nl/min using the following gradient: 2% B (from 0–5 min), and 2–70% B (from 5–55 min). Solvent A consisted of 0.05% formic acid in 98% H2O/2% ACN and solvent B consisted of 0.05% formic acid in 98% ACN/2% H2O. The column eluant was directly coupled to a QSTAR Pulsar i quadrupole orthogonal TOF mass spectrometer (MDS Sciex, Concorde, Canada) equipped with a Protana/ProXeon nanospray ion source (ProXeon Biosystems, Odense, Denmark). The nanospray needle voltage was typically 2,300 V in the HPLC-MS mode. Mass spectra (ESI-MS) and tandem mass spectra (ESI-MS/MS) were recorded in positive-ion mode with a resolution of 12,000–15,000 FWHM. For CID-MS/MS, the mass window for precursor ion selection of the quadrupole mass analyzer was set to ±1 m/z. The precursor ions were fragmented in a collision cell using nitrogen as the collision gas. All ESI-MS and MS/MS spectra were externally calibrated in static nanospray mode using MS/MS fragment ions of a renin peptide standard (His immonium ion with m/z at 110.0713, and b8 ion with m/z at 1028.5312) providing a mass accuracy of ≤50 ppm. For a selected set of samples (immunoprecipitated Complex I from mouse brain), the acquired LC-MS datasets were further calibrated applying an internal "one-point" calibration on top of the original external "two-point" calibration to achieve an even higher mass accuracy (≤20–30 ppm). In each case, a peptide mass from the ESI-MS trace was selected for internal calibration of a particular LC-MS run after an initial Mascot database search identified a protein match and assigned corresponding peptides. Only peptides that were previously selected for MS/MS displaying confident fragmentation patterns were used for recalibration of the entire LC-MS file. Due to the nature of a "one-point" calibration the slope of the calibration curve stayed unchanged, whereas the intercept changed slightly.

Database Searches—
Mass spectrometric data were analyzed with two in-house licensed bioinformatics database search engine systems, RADARS (Genomic Solutions, Ann Arbor, MI) (45) and Mascot (Matrix Sciences, London, United Kingdom) (46). MALDI-MS data were analyzed with RADARS using the search engine ProFound for PMF matching against peptides from known protein sequences entered in publicly available protein databases (e.g. NCBI) using the following parameters: internal calibration using trypsin autolysis masses (m/z 842.5100 and 2211.1046), 100 ppm mass accuracy, two missed proteolytic cleavages allowed. Profound uses an "expectation value" for data quality control that gets smaller as the probability of a nonrandom (real) protein hit increases, e.g. 1 x 10–2 is a 1 in 100 chance of being a random hit (confidence > 99.0%), 1 x 10–3 is a 1 in 1,000 chance of being a random hit (confidence > 99.9%); protein matches are considered significant for scores with expectation value <5 x 10–2 (confidence > 95%) (45). In all cases, tryptic digestion extracts of proteins were also analyzed by HPLC-ESI-MS and MS/MS. For ESI-MS/MS datasets, spectra were submitted to Mascot. Custom-designed databases for bovine, mouse, and rat Complex I were incorporated into Mascot to enable more in depth searches (for accession numbers see Table S1, Supplement). Mascot uses a probability based "Mowse Score" to evaluate data obtained from tandem mass spectra, e.g. for a score >37, protein matches are considered significant (46). For LC-MS/MS-acquired data, a minimum of two observed peptides that were selected for MS/MS was required to confirm protein identification; in the few cases where only one peptide per protein was selected for MS/MS, the MS/MS spectrum was inspected "manually" and thus confirmed or deleted from the identification list. Last, to extract and compile peak lists from ESI-MS data generated during an LC-MS/MS run, Mascot Distiller (1.1.1) and Distiller MDRO Software Developer’s Package (Matrix Sciences) were used in conjunction with an in-house Java program, "MS-Assign." To reduce false positives and eliminate noise spikes, an exclusion parameter was implemented that required mass peaks (with identical m/z) to appear in at least two adjacent ESI-MS survey-scans. Confident peak picking was established and confirmed by extensive checking of peak picking mass list outputs against the raw data through verification of isotope patterns, charge states, and peak shapes. Subsequently, these ESI-MS peak lists were submitted to the Mascot search engine for PMF analysis at a mass accuracy of ±30 ppm. To display this data, a Java program was developed, "SeqDisp," to graphically display the observed protein sequence coverage of peptides obtained from both ESI-MS/MS and from ESI-MS data, as well as calculating the overall protein coverage.

Complex 1 Databases and Blast Search/Sequence Alignment Tools—
Custom-designed protein databases of mouse and rat Complex I proteins were constructed so that MS and MS/MS data could be searched more exhaustively by RADARS and Mascot for better sequence coverage and identification (for a complete list of accession numbers for mouse and rat Complex I databases see Supplemental Table S1). The NCBI Blast Search algorithm was used to construct mouse- and rat-specific Complex I databases; we previously have assembled a bovine Complex I database and could use that latter database for comparison. Initial sequence alignment calculations were performed with the program "SIM Alignment Tool" (47) using the following parameters: comparison matrix, BLOSUM62; number of alignments computed, 20; gap open penalty, 12; gap extension penalty, 4. The program "ClustalW" (at EMBnet-CH) for Mulitiple Sequence Alignment was run to do extensive comparisons of Complex I subunits across several species (48).


    RESULTS
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In the present study, we have used mouse heart and brain tissues, and the rat dopaminergic neuronal cell line N27, to develop two independent one-step purification methods of intact mitochondrial Complex I. Mitochondria were isolated and solubilized (40), and Complex I was isolated by sucrose gradient fractionation (method A) or direct immunoprecipitation (method B). Intact and functional Complex I that was obtained by these methods was further separated by BN-PAGE, 1D SDS-PAGE, and/or 2D SDS-PAGE and then characterized by MS.

Sucrose Gradient Fractionation of Mouse Brain Complex I—
Complex I was first isolated from rodent neuronal samples using a sucrose gradient fractionation procedure originally described by Hanson et al. (27) and then used in our own studies to define the human heart mitochondrial proteome (28, 49). Mouse brain mitochondria were first solubilized with 1% maltoside and fractionated on a 10–35% sucrose gradient. Aliquots of the sucrose gradient fractions were collected and run on a 10–20% SDS-PAGE gel and probed with anti-Complex I mAb (39 kDa) as shown in Fig. 1A. Although traces of Complex I were found in the pellet fraction (Fig. 1A, lanes 1 and 2), the sucrose gradient fractions 4–10 (Fig. 1A, lanes 4–10) were highly enriched in Complex I based on Western staining, and those fractions were pooled and concentrated. An aliquot of the pooled fractions 4–10 was run on a nondenaturing 4–15% gradient BN-PAGE gel (see Fig. 1B, lane 1). As expected, intact Complex I migrated at an estimated molecular mass of ~700 kDa and was found to be the major component of the selected/pooled sucrose gradient fractions. Although the theoretical molecular mass of Complex I was reported as 980 kDa (for bovine Complex I) (23), it was previously shown to migrate at a slightly lower Mr by BN PAGE (50). To confirm the identity of Complex I subunits, the gel bands were excised, proteolyzed with trypsin, and analyzed by HPLC-ESI-MS/MS on a hybrid Q-TOF mass spectrometer (QSTAR). A single LC-MS/MS run produced sufficient MS/MS data to identify 24 subunits of mouse brain Complex I. When this dataset was searched against our restricted mouse Complex I database, an additional eight Complex I subunits were identified, yielding a total of 32 out of the expected 45 subunits from the BN-PAGE-purified material. Two other weakly stained BN gel bands were also analyzed and identified as Complex III and V, co-migrating at ~ 450 kDa (Complex III migrates as dimer as reported by Schagger et al. (41, 42)), and Complex IV, migrating at ~ 200 kDa as annotated in Fig. 1B, lane 1.



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FIG. 1. Purification of mouse brain mitochondrial Complex I by sucrose gradient centrifugation analysis. Mouse brain mitochondria were solubilized with 1% maltoside and fractionated on a 10–35% sucrose step gradient (38,000 rpm/16.5 h). A, aliquots (20% of volume) of the sucrose gradient fractions were run on 10–20% SDS-PAGE gels followed by Western blot analysis with anti-Complex I (39-kDa) mAb (lanes 3–15). Lanes 1 and 2 correspond to pellet fraction, the lane marked input contains unfractionated mitochondrial extract. Complex I-positive sucrose gradient fractions 4–10 (lanes 4–10) were pooled. B, subsequently, the pooled fraction was separated on a nondenaturing 4–15% Blue Native Tris PAGE gel (20% of pooled fraction volume was loaded) and stained with Coomassie Brilliant Blue G-250 (lane 1). Lane STD shows molecular mass standards. Complex I was identified to be the major component of the selected sucrose gradient fractions and migrated at an estimated molecular mass of 700 kDa. Complexes III and V (migration at 450 kDa; Complex III migrates as dimer as reported by Schagger et al. (41, 42)) and Complex IV (migration at 200 kDa) were identified as low-abundant side products.

 
In a second experiment, the pooled sucrose gradient fractions containing Complex I were analyzed by 1D and 2D SDS gel electrophoresis as described earlier (28). An aliquot of the pooled sucrose gradient fraction was denatured, loaded onto a 10–20% 1D SDS-PAGE, and stained with Sypro Ruby (see Fig. 2). To obtain peptide sequence data, the protein bands were excised from the 1D SDS gel shown in Fig. 2, digested with trypsin, and analyzed by MALDI-TOF-MS prior to analysis by HPLC-ESI-MS/MS. Mass spectrometric analysis and data processing identified 34 out of 45 rodent Complex I subunits as annotated for the bands in Fig. 2. The mass spectrometric details of this protein identification are listed in Table I. As shown in Fig. S1, enriched mouse brain Complex I obtained from sucrose gradient fractionation was also separated by 2D SDS-PAGE (see Supplement).



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FIG. 2. Mouse brain mitochondrial Complex I purified by sucrose gradient fractionation and separated into its subunits by 10–20% Tris SDS-PAGE. Twenty percent of total volume of the combined, concentrated sucrose gradient fractions (that were highly enriched in Complex I) was loaded on the gel. The Complex I lane was stained with Sypro Ruby, and visualized bands were excised from the gel and subjected to in-gel digestion. Mass spectrometric analysis identified 34 out of 45 Complex I subunits as annotated for each band.

 

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TABLE I Complex I subunits (mature proteins) from mouse brain identified by ESI-MS/MS from sucrose gradient/1D SDS gel and IP/1D SDS gel preparation

 
At this point, samples obtained from sucrose gradient purification yielded good coverage for a majority of the subunits when subjected to MALDI-MS and/or HPLC-MS/MS analysis after additional BN-PAGE or 1D and 2D SDS-PAGE separation. Although HPLC-ESI-MS/MS provided the best sequence coverage, even peptide mass fingerprint data acquired by MALDI-TOF was sufficient in many cases to confidently identify the Complex I subunits. For example, Fig. 3 shows two MALDI-MS spectra recorded from the tryptic digestions of gels spots obtained after 2D gel separation of the pooled sucrose gradient fractions enriched in Complex I from mouse brain mitochondria. Both proteins were clearly identified as Complex I subunits NDUFS3 (30-kDa subunit, see Fig. 3A) and NDUFS6 (13-kDa subunit, see Fig. 3B), respectively. For NDUFS3 (Fig. 3A), eight tryptic peptides were assigned by "Profound," yielding a sequence coverage of 28% (63/228 amino acids) and a score of 1.8 x 10–3 (for details see "Experimental Procedures"). Similarly, five peptide masses could be assigned to the NDUFS6 (Fig. 3B) for a total sequence coverage of 69% (65/96 amino acids) and score of 3.9 x 10–5. Of particular interest were tryptic peptides containing cysteine residues, such as those from NDUFS6 at m/z 1391.6 (M = 1390.6, TGTC*GYC*GLQFK, residues 81–92) and m/z 1422.7 (M = 1421.7, IIAC*DGGGGALGHPK, residues 56–70).



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FIG. 3. MALDI-TOF mass spectra of Complex I subunits NDUFS3 (A) and NDUFS6 (B) isolated from sucrose gradient-purified Complex I from mouse brain. The MALDI-MS PMF spectra display molecular ions of peptides obtained after in-gel tryptic digestion of the selected protein spots from a 2D SDS-PAGE gel. The observed masses are labeled and annotated with starting and ending amino acids. A shows eight-peptide data resulting from tryptic digestion of Complex I subunit NDUFS3 (30-kDa subunit, NUGM). Overall, a protein sequence coverage of 28% was observed for the mature 26.5-kDa protein after removal of the mitochondrial import sequence; theoretical pI of 5.5 (63/228 amino acids). B shows five PMFs resulting from tryptic digestion of mature Complex I subunit NDUFS6 (13-kDa subunit, NUMM). Overall, a protein sequence coverage of 69% was observed for the mature 10.8-kDa protein; theoretical pI of 6.6 (65/96 amino acids). T, trypsin autolysis peptides; C*, cysteine residue alkylation with iodoacetamide.

 
In total, by combining the results from the sucrose gradient purification of mouse brain mitochondria followed by BN-PAGE, 1D, or 2D SDS-PAGE, 37 out of 45 subunits of Complex I were identified (see Fig. 4 and Table I). The mass spectrometric details of the Complex I subunit identification using sucrose gradient purification are presented in Table I, including protein sequence coverage and the number of peptides identified by MS/MS. The latter information provides some qualitative information on the confidence of the protein assignments and, more importantly, is used to provide statistical scores using the MOWSE score for MS/MS sequence data searches (46).



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FIG. 4. Overview and comparison of two independent one-step purification methods, sucrose gradient fractionation and IP of rodent mitochondrial Complex I. Complex I was isolated from mouse brain mitochondria and from mitochondria obtained from a rat N27 dopaminergic neuron cell line, respectively, either by sucrose gradient fractionation and/or by IP with mAb 18G12. Subsequently, the Complex I preparation was further separated by BN-PAGE or by SDS-PAGE (1D and 2D gel electrophoresis analysis), and subjected to mass spectrometric characterization. In addition, combinations of these two independent one-step purification methods were performed, including sucrose gradient fractionation and subsequent IP of Complex I from enriched sucrose gradient fractions. #, gel slices were digested (in-gel) and analyzed by MALDI-MS and HPLC-ESI-MS/MS; *, database searches were performed using the search engines ProFound/RADARS (Genomic Solutions) and Mascot (Matrix Science) against custom-designed rodent Complex I databases.

 
Immunocapture of Complex I from Mouse Brain and Heart Mitochondria—
Previously, Murray et al. (29) developed mAbs for the immunocapture of bovine Complex I that were also effective against Complex I from human heart mitochondria. We now optimized the efficiency of these immunocapture antibodies through the construction of a "second generation" mAb set (51). To evaluate the efficiency of these newer mAbs for the IP and purification of Complex I from rodent and other species, both analytical and immunocapture antibodies (anti-bovine Complex I mAb) were tested against mitochondria obtained from mouse. First, two 1D SDS-PAGE gels (10–22% Tris-HCl acrylamide gels) were run in parallel, loaded with either 50 µg of bovine heart mitochondria or mouse heart mitochondria. The proteins were then transferred to the PVDF membrane and probed by Western blot with a mixture of eight analytical Complex I antibodies (for details see "Experimental Procedures"). Two of the eight analytical antibodies (mAbs 8K 17C8 and S5 21A6) showed slightly less affinity for the mouse samples (compared with the bovine samples), but in all other cases the analytical mAb showed identical specificity (positive recognition) for mouse and bovine samples (data not shown). Second, five mAbs were tested for their immunocapture abilities of Complex I across species. The results for the two most-efficient mAb are shown in Fig. S2 (see Supplement). Briefly, bovine heart mitochondria (lanes 1 and 2, 100 µg each) and mouse heart mitochondria (lanes 3 and 4, 100 µg each) were incubated with the different Complex I immunocapture antibodies mAb 20D1 and mAb 18G12; the resulting immunoprecipitates were then separated by 1D SDS-PAGE. Antibodies mAb 20D1 and mAb 18G12 showed successful immunocapture of mouse Complex I, and an almost identical pattern of Complex I subunits was observed for bovine and mouse samples after separation by 1D SDS- PAGE (see Fig. S2, Supplement). After these successful pilot experiments, mAb 18G12 was selected for all further experiments with mouse and rat mitochondria obtained from rodent tissue and rodent cell lines.

For in-depth characterization, intact rodent Complex I was isolated by immunopurification from mouse heart and brain mitochondria using mAb 18G12 and subsequently separated by 1D SDS-PAGE as shown in Fig. 5. All major 1D SDS gel bands obtained after the IP experiment from mouse heart (lane 1) and mouse brain mitochondria (lane 2), respectively, were assigned to Complex I subunits. For the complete proteomic characterization of immunopurified Complex I from mouse heart and mouse brain mitochondria after 1D SDS-PAGE separation, we used a combination of MALDI-MS PMF techniques and a more in-depth online nano-HPLC-ESI-MS/MS analysis. These data are summarized in Table I. Overall, 41 out of a total of 45 Complex I subunits were identified from this IP/1D SDS-PAGE experiment as annotated for each band in Fig. 5.



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FIG. 5. Immunopurified mouse complex I separated by 1D SDS-PAGE. Complex I was immunopurified from mouse heart (40 µg) and brain (300 µg) mitochondria using mAb 18G12 and subsequently resolved on a NuPAGE 4–12% BisTris (MES) gel. Lane 1 shows Complex I purified from mouse heart mitochondria; lane 2 shows Complex I purified from mouse brain mitochondria. The lanes were stained with Coomassie Brilliant Blue R. Bands were excised from the gel and proteolytically digested. Mass spectrometric analysis identified 41 out of 45 Complex I subunits as annotated for each band.

 
The IP of mouse Complex I and the separation of its subunits by 1D SDS-PAGE analysis significantly decreased the sample complexity and allowed for a better overall characterization. Several Complex I subunits (>10 proteins) could be readily assigned by rapid MALDI-MS PMF screening alone, including NDUFS1 (32% coverage), NDUFA9 (26% coverage), NDUFA2 (37%), NDUFS2 (26%), NDUFA8 (47%), and NDUFB4 (61%), to name a few (data not shown). To achieve a more in-depth characterization of mouse Complex I subunits, each 1D SDS-PAGE gel band obtained from the immunopurification experiment shown in Fig. 5 was subjected to LC-MS/MS analysis. In some cases, the 1D SDS gel bands contained protein mixtures of up to six subunits from Complex I (e.g. see Fig. 5, gel band 13). Therefore, these 1D gel bands required further separation using online reversed-phase nano-HPLC-MS/MS. Database search results for the obtained MS/MS data obtained from the IP experiment were performed using our in-house search engine Mascot (Matrix Science) as displayed in Table I (see "ESI-MS/MS sequence coverage" column below IP heading). For the MS/MS data, we also report the number of observed peptides selected for MS/MS and the corresponding total Mascot score. A representative MS/MS spectrum from this dataset is shown in Supplementary Fig. S3 corresponding to the peptide V65VAAC*AMPVMK75 from Complex I subunit NDUFS1. Similarly, tandem mass spectra were recorded for the remaining mouse brain Complex I protein subunits obtained after IP with mAb 18G12 and 1D gel separation.

To increase the coverage of Complex I subunits, we developed a strategy to extract the peptide mass data from HPLC-ESI-MS/MS runs. Mascot Distiller (1.1.1) and Distiller MDRO Software Developer’s Package were used in combination with an in-house-developed Java program, "MS-Assign," to extract and compile ESI-MS peak lists from the LC-MS/MS runs (see "Experimental Procedures" for details). Prior to generating the ESI-MS peak lists, the selected LC-MS files were internally recalibrated applying a "one-point" calibration on top of the original external "two-point" calibration to achieve an even higher mass accuracy. PMF lists (ESI-MS peak lists) generated using the programs Mascot Distiller and "MS-Assign" were then subjected to database searches against our in-house Mascot server (30 ppm mass accuracy). Having a custom-designed mouse Complex I database available (see "Experimental Procedures" and Supplementary Table S1) was of particular advantage for performing these ESI-PMF searches as such searches could be quite time-intensive otherwise. Resulting Complex I subunit PMF sequence coverage is reported in Table I (under "Sequence cov. ESI-MS PMF"). As shown in Table I, the protein sequence coverage for many Complex I subunits were already quite high based on ESI-MS/MS data alone (as high as 83%), but were significantly improved when a PMF search (ESI-MS data) was carried out against all tryptic peptide masses assignable when searched against the Complex I database. Typically, additional sequence coverage of between 10–30% was achieved for most subunits. Such a restricted search is possible only due to the high purity of the isolated protein complex.

The IP protocol clearly achieved a superior resolution of the Complex I subunits, and resulted in a higher sequence coverage than the sucrose gradient method. All of the major bands visualized by 1D SDS-PAGE were Complex I subunits. However, some "coprecipitating/interacting proteins" were also identified at low level, in several cases from regions of the gel where almost no protein stain was observed. Most of these low-abundance co-precipitating proteins are subunits from other ETC complexes (Complexes II–V) and may, in fact, be components of so-called supercomplexes (50, 52). These proteins included one subunit of Complex II, four subunits of Complex III, six subunits of Complex IV, six subunits of Complex V. A complete list of "co-precipitating/interacting proteins" is given in Supplemental Table S2.

As a control and for comparison purposes, bovine Complex I was also immunoprecipitated with this second generation mAb 18G12 from bovine heart (as indicated above), resolved by 1D SDS-PAGE, and subsequently subjected to mass spectrometric analysis, which gave very comparable results (see Supplemental Fig. S4 and Table S3).

Immunocapture of Complex I from N27 Neuronal Rat Cells—
Given the success of Complex I immunocapture from mouse brain, a rat N27 dopaminergic cell line was examined using a similar protocol. Compared with heart muscle or even brain tissue, the rat N27 dopaminergic cell line contains a significantly lower amount of mitochondria. In Fig. 6A, a 1D SDS-PAGE separation is shown of immunocaptured Complex I from isolated mitochondria (200 µg total protein) obtained from the N27 cell line using the same mAb used in the preceding mouse experiments, mAb 18G12. The protein banding pattern obtained in this gel separation is nearly identical to the profile obtained for comparable Complex I preparations from mouse heart (lane 1, 40 µg) and brain (lane 2, 300 µg). However, a reduced intensity for the resolved protein subunits was observed in the rat cell line compared with the other two sources. To obtain the best possible staining sensitivity, the gel was restained with silver (53) (lanes 1 and 2 appear overexposed using the silver stain to visualize lane 3). For mass spectrometric analysis, the gel was rerun and stained with Coomassie Brilliant Blue, and bands were excised by complete "rastering" (from top to bottom) of the gel lane, as only "landmark subunits" were visible with Coomassie Brilliant Blue. All samples were digested with trypsin and analyzed by LC-MS, MS/MS. As summarized previously in Fig. 4, 33 out of 45 Complex I subunits were identified (see Table II).



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FIG. 6. Immunopurified dopaminergic rat cell line (N27) Complex I separated by 1D SDS- PAGE. A, Complex I was immunopurified from 200 µg of dopaminergic neuronal mitochondria (rat N27 cell line) as shown in lane 3 using mAb 18G12. For comparison, lane 1 displays immunopurified mouse heart Complex I (from 40 µg of mitochondria) and lane 2 displays immunopurified mouse brain complex I (from 300 µg of mitochondria). The Tris-HCl 10–22% acrylamide gel shown in A (lanes 1–3) was stained with silver stain according to the protocol of Shevchenko et al. (53). Lanes 1 and 2 appear overexposed in order to visualize lane 3 containing Complex I from the dopaminergic rat cell line. B shows a different Sypro Ruby-stained gel (NuPAGE 4–12% BisTris (MES) gel) obtained from a different experiment in which a total of 1 mg of dopaminergic neuronal mitochondria (rat N27 cell line) were first separated by sucrose gradient fractionation; subsequently, the sucrose gradient fractions that were enriched in Complex I were then subjected the to the Complex I immunocapture protocol.

 

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TABLE II Complex I subunits, rat cell line N27, mature/processed proteins identified by ESI-MS/MS from IP/1D SDS gel preparation

 
Because the total mitochondria in the rat neuronal cell line is considerably lower than mouse brain, an additional protocol was developed to increase Complex I immunocapture efficiency. Rat N27 cell line mitochondria (1 mg) were first separated by sucrose gradient fractionation, and the fractions enriched in Complex I were pooled and dialyzed against the immunocapture buffer and then incubated with mAb 18G12. In this experiment (Fig. 6B, lane 4) the 1D SDS-PAGE gel was stained with Sypro Ruby and the rat Complex I subunits were identified by mass spectrometric analysis similar to the procedure described above.


    DISCUSSION
 TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Mitochondrial Complex I plays a key role in cellular metabolic processes, and many mitochondrial diseases and neurological disorders are linked to loss of Complex I activity. Studies using samples from PD patients and animal and cell culture models of the disease have clearly indicated a correlation between Complex I inhibition and PD pathology. Therefore, to provide a means to isolate and characterize Complex I in rodent models of PD, we developed two independent and rapid experimental strategies to efficiently isolate OXPHOS Complex I from several rodent mitochondrial sources using sucrose gradient fractionation and IP.

Both protocols can be used with small amounts of starting material and are capable of rapidly isolating Complex I from a variety of tissues. As summarized previously in Fig. 4, these independent one-step purification methods consist of sucrose gradient fractionation or the direct immunocapture of rodent Complex I from mouse tissues, as well as a rat cell line (N27), mitochondria. Subsequent analyses of these preparations were carried out after separation by BN and/or 1D or 2D SDS-PAGE. Using sucrose gradient fractionation of mouse brain mitochondrial proteins, a relative pure preparation was obtained with only trace contamination from Complexes III, IV, and V. Mass spectrometry sequence analysis of this purified material led to the identification of 37 out of the 45 subunits of Complex I. The direct immunocapture method of Complex I from mouse brain employing the new anti-Complex I mAb (18G12) increased the total number of subunits identified by MS/MS to 41 out of 45. The rodent protein Complex I subunits that were not identified in this latter analysis were subunits known to be highly problematic, i.e. ND2, ND6, ND4L, and NDUFC1. Mouse subunit ND6, for example, does not generate any tryptic peptides within the investigated mass range (m/z 700–4000) and mouse subunit ND4L is highly hydrophobic and only generates two single large tryptic peptides. Subunit NDUFC1 (6-kDa subunit) is a very small subunit (in fact, the smallest in Complex I). For the bovine control, only two of the 45 subunits were not identified, i.e. ND6 and ND4L (see Supplemental Table S3), and this high coverage allowed us to identify several PTMs (data not shown). Even for the rat cell line that yields significantly less mitochondria, 33 of 45 subunits were readily assigned by MS/MS. In cases such as these, sucrose gradient fractionation prior to IP may be a preferable strategy to increase coverage.

A sequence comparison of Complex I subunits among species (bovine, mouse, and rat) shows very high identity and homology. Bovine and mouse Complex I, for example, show sequence identities greater than 80% for 27 subunits. Likewise, bovine and rat Complex I contain 26 subunits with this same level of identity (for details see Supplemental Table S1). To further emphasize the high sequence identity of Complex I proteins between the species also see Supplemental Fig. S5 showing a comparison of MS/MS spectra of homologous peptides obtained from bovine and rat Complex I subunit B16.6/Grim 19. A comparison among species of all cysteine residues (~120 residues) within the 45 subunit shows that this is a highly conserved amino acid; between mouse and human Complex I subunits, 29 of 45 subunits show identical numbers of conserved cysteines and an additional 13 subunits differ by only one cysteine. The rapid purification methods we have described here are likely to limit the introduction of artifacts during Complex I isolation and would be highly suitable to study protein oxidation or other types of protein modifications that might accompany disease and aging processes.

To investigate oxidative stress of the Complex I protein subunits, we propose to use this new methodology to thoroughly analyze ESI-MS and MS/MS datasets from several rodent models of the disease to achieve optimal protein mapping and sequence coverages. For this purpose, a Java program named "SeqDisp" was developed in-house that graphically displays the protein sequence coverage of peptides obtained from both ESI-MS and MS/MS datasets as well as calculating the coverage and counting for specific amino acid residues, such as cysteines. An example is shown in Supplemental Fig. S6, graphically showing the sequence coverage increase for Complex I subunit NDUFS1 from 55% (8 of 18 Cys observed) to 62% (12 of 18 Cys observed) when considering additional ESI-MS data and not solely the ESI-MS/MS data.

In addition, we (18, 19) and others (20, 21) have recently developed stable isotope differential cysteine alkylation methods to label cysteine residues in proteins as a means to assess cysteine redox status. Of particular interest would be to investigate reversible and nonreversible cysteine oxidation of Complex I in the glutathione-depleted rat dopaminergic N27 cell lines (a PD model) and to identify protein subunits and key cysteine residues that may undergo PTMs. Previous work by Walker’s group in Cambridge has shown numerous PTMs in bovine Complex I (23, 36), but these have yet to be correlated to any disease or functional model. We observed similar PTMs in Complex I obtained from mouse brain including N-terminal acetylation of several subunits (NDUFA2, NDUFA6, NDUFB6, NDUFB9, and B17.2), as well as several nonenzymatic modifications, such as deamidation of Asn, oxidation of Trp to formylkynurenine, and oxidation of Met to the sulfone and sulfoxide. However, these latter nonenzymatic PTMs need to be evaluated more carefully to establish any biological significance as they can easily be introduced in sample isolation and work-up protocols. Future work will attempt to exploit this rapid method for Complex I purification to identify PTMs, such as phosphorylation, nitration, or other oxidative events that may be involved in aging or disease processes. This current study provides us with a set of tools to investigate Complex I-related diseases, such as PD, in detail in rodent disease models.


    ACKNOWLEDGMENTS
 
We thank Chip Witt (Buck Institute) and John Cottrell (Matrix Sciences) for helpful bioinformatics advice.


    FOOTNOTES
 
Received, September 30, 2004, and in revised form, December 10, 2004.

Published, MCP Papers in Press, December 10, 2004, DOI 10.1074/mcp.M400143-MCP200

1 The abbreviations used are: PD, Parkinson’s disease; ETC, electron transport chain; SN, substantia nigra; ROS, reactive oxygen species; GSH, glutathione; IP, immunoprecipitation; mAb, monoclonal antibody; RNS, reactive nitrogen species; PTM, posttranslational modification; N27, rat dopaminergic neuronal cell line 1RB3AN27; PMF, peptide mass fingerprint; 1D, one dimensional; 2D, two dimensional; BN, Blue Native. Back

* This work was supported by National Institutes of Health Grant R21 NS043620-01 (to B. W. G.). S. B. M.M. is a recipient of a postdoctoral fellowship from the National Parkinson Foundation-Parkinson Disease Foundation Joint Program. The costs of publication of this article were defrayed in part by the pay-ment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

S The on-line version of this manuscript (available at http://www.mcponline.org) contains supplemental material. Back

§ B. S. and S. B. M.M. contributed equally to this work. Back

§§ To whom correspondence should be addressed: Buck Institute for Age Research, 8001 Redwood Boulevard, Novato, CA 94945. Tel.: 415-209-2032; Fax: 415-209-2231; E-mail: bgibson{at}buckinstitute.org


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