Cysteine-reactive maleimide-conjugated Cy3/5 cyanine Ettan DIGE Saturation Labeling dyes (Cys-CyDyes) and lysine-reactive N-hydroxysuccinimide (NHS) ester-conjugated Cy3/5 cyanine Ettan DIGE Minimal Labeling dyes (Lys-CyDyes) were purchased from GE Healthcare (Piscataway, NJ). Sequencing Grade Modified Trypsin and Gold Mass Spectrometry Grade Modified Trypsin were purchased from Promega (Madison, WI). Solutions and stocks for in-gel trypsin digest and mass spectrometry procedures were prepared using HPLC-grade ddH2O from Fisher Biotech (Pittsburgh, PA) and HPLC-grade MeOH and acetonitrile from Sigma-Aldrich (St. Louis, MO). Protease inhibitor cocktail (cat#P2714), DA, mushroom tyrosinase, and most general chemicals for SDS-PAGE, buffers, and solutions were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. Rabbit-anti-mitochondrial creatine kinase (MtCK) and rabbit-anti-mitofilin polyclonal antibodies were generated for our laboratory by GeneMed Synthesis, Inc. (South San Francisco, CA). All other general solutions and stocks were prepared using doubly distilled water (ddH2O) from a Milli-Q system (Millipore Corp., Bedford, MA).
Mitochondrial Isolation and Respiration
All animal procedures were approved by the Animal Care and Use Committee at the University of Pittsburgh and are in accordance with guidelines put forth by the National Institutes of Health in the Guide for the Care and Use of Laboratory Animals
. Mitochondria-enriched fractions were isolated from the brain tissue of adult male Sprague-Dawley rats via differential centrifugation by the method of Rosenthal et al. (1987)
as previously described (Berman and Hastings, 1999
; Berman et al., 2000
), with elimination of the protease Nagarse. Final mitochondrial pellets were resuspended in mitochondrial isolation buffer (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mg/ml FA-free BSA, and 1 mM EGTA, pH 7.4) and kept on ice. Mitochondrial protein content was determined by the Bradford method (1976)
. Prior to experimental use, respiration rates based on oxygen consumption were measured in the mitochondrial preparations to ensure mitochondrial health, as previously described (Berman and Hastings, 1999
). Mitochondrial health was determined by the ratio of respiration active state 3, induced by the addition of ADP, to resting state 4, induced by the addition of oligomycin. Only mitochondria with a coupled state 3/state 4 ratio above 6 were used for this study.
Exposure of Isolated Mitochondria to Dopamine Quinone
Mitochondria (2 mg total protein) were exposed to DA (150 µM) and tyrosinase (150 U) in modified mitochondrial isolation buffer with 25 mM HEPES minus BSA, pH 7.4 (reaction buffer) plus protease inhibitor cocktail (2.5 µl/mg protein; Sigma) for 15 min at room temperature (RT). Following incubation, mitochondria were placed on ice and immediately pelleted by centrifugation at 15,000 × g for 15 min at 3°C. Control mitochondria underwent the same procedure in the absence of DA. Pelleted mitochondria were lysed by rigorous pipetting in denaturing 2-D DIGE lysis buffer (9 M urea, 2% w/v CHAPS, and 30 mM Tris-base, pH 8.5) in a ratio of 100 µL buffer to 1 mg protein. Insoluble material was pelleted by centrifugation (16,000 × g for 1–2 min at RT) and discarded. Protein concentrations of lysed control and DAQ-exposed samples were determined by Bradford (1976)
. Thiol reducing agents were excluded from the lysis buffer to maintain proteins in a non-reduced state.
Cys- and Lys-CyDye Labeling
For cysteine-dye minimal labeling 2-D DIGE, migration-matched Cy3 and Cy5 Cys-CyDyes (GE Healthcare) were rehydrated in dimethylformamide (DMF) to a concentration of 0.5 mM, aliquoted, and stored at −20°C with desiccation until use. Prior to use, an aliquot of dye was thawed to RT and diluted in DMF to a working concentration of 62.5 µM. Control and DAQ-exposed protein sample lysates were reacted with the indicated Cys-CyDye under non-reducing conditions at a ratio of 1 pmol dye per 2 µg protein. We used low concentrations of Cys-CyDyes to achieve a minimal labeling effect on non-reduced protein samples. Preliminary experiments using various dye concentrations identified 1pmol dye per 2 µg protein, which is 0.125% of the ratio utilized for saturation labeling, as optimal for minimal Cys-CyDye labeling. This concentration provided sufficient labeling for detection and imaging while maintaining reproducible results across gels (data not shown).
Samples were labeled Cys-Cy5 control and Cys-Cy3 DAQ, or the reciprocal to control for differential dye affinity. Samples were gently vortexed and incubated in the dark for 45 min at RT. The reaction was quenched by adding an equal volume amount of 2-D DIGE sample buffer (9 M urea, 2% w/v CHAPS, 2% v/v 3–10 IPG ampholyte buffer, 130 mM dithiothreitol (DTT), and a trace of bromophenol blue in ddH2O). Final DIGE samples were prepared by combining equal amounts of Cys-CyDye labeled control protein and Cys-CyDye labeled DAQ-exposed protein.
Lysine-dye minimal-labeling 2-D DIGE analysis was utilized to control for changes in protein abundance between control and DAQ-exposure groups in comparison to Cys-CyDye DIGE. Migration-matched Cy3 and Cy5 Lys-CyDyes were rehydrated in DMF to a concentration of 0.5 mM and stored at −20°C with desiccation until use. Prior to use, dyes were thawed to RT and diluted 1:1 in DMF. Control and DAQ-exposed protein sample lysates were reacted with the indicated Lys-CyDye (Lys-Cy5 control and Lys-Cy3 DAQ, or the reciprocal) under non-reducing conditions at a ratio of 2 pmol dye per 1µg protein in the dark for 30 min on ice. The reaction was quenched by the addition of free lysine to a final concentration of 385 µM and incubated 15 min on ice. Labeled samples were diluted 1:1 with 2-D DIGE sample buffer. Equal protein amounts of the Lys-CyDye labeled control and the DAQ-exposed samples were combined to generate a final DIGE sample for 2-D gel electrophoresis. Each DIGE gel experiment and its associated parallel gels (Cys- and Lys-CyDye DIGE gels and reciprocals) were generated from independent mitochondrial isolation and DAQ exposure experiments.
2-Dimension Difference In-Gel Electrophoresis
Samples (250 µg) were isoelectrically focused via sample cup loading on rehydrated 18 cm linear 3–10 pH Immobiline DryStrips (GE Healthcare), using a Multiphor II system with a 3501XL power supply (GE Healthcare), and using a 4-phase program with a total run of 75 kVhr. Focused strips were stored at −80°C until the second dimension run. Prior to second dimension electrophoresis, DryStrips were equilibrated at RT for 10 min in an equilibration buffer (75 mM Tris-HCl pH 6.8, 6 M urea, 30% v/v glycerol, 1% w/v SDS) supplemented with 30 mM DTT, followed by 10 min at RT in equilibration buffer supplemented with 240 mM iodoacetamide. Equilibrated DryStrips were trimmed to 13.5–15 cm and run in second dimension 12% SDS-PAGE (1.5mm thick gels, Hoefer SE600 Ruby Electrophoresis Unit).
Fluorescence Detection and Spot Picking
Immediately following the second dimension run, gels were scanned on a Typhoon 9400 scanner using ImageQuant software (GE Healthcare) to obtain a 200 µm resolution image of the gel. Following imaging, gels were fixed overnight in a 40% MeOH, 1% acetic acid solution at 4°C. The gels were scanned a second time using a fluorescent scanning automated spot picker, designed by Dr. Jonathan Minden of Carnegie Mellon University (instrumentation housed in the University of Pittsburgh Genomics and Proteomics Core Laboratories). The digital scans of the Cy3 and Cy5 dyes within each gel were compared visually with the aid of Image J imaging software (NIH). Protein spots that exhibited a noticeable change in fluorescence, and several that exhibited no change, were then picked utilizing the automated picker.
In-gel Trypsin Digest and Protein Identification
Immediately following excision from 2-D DIGE gels, gel plugs were washed with 50:50 MeOH:50 mM ammonium bicarbonate followed by dehydration in acetonitrile (ACN) and drying by speed-vacuum. The dried plugs were rehydrated with 10 µl of 20 µg/ml trypsin in 20 mM ammonium bicarbonate and then incubated for 4 hr at 42°C. Samples were extracted by repeated washing in 1% trifluoroacetic acid in 50:50 ACN:H2O extraction buffer, dried completely via speed-vacuum, and stored for up to 2 weeks with desiccation at 4°C.
Protein identification was completed using MALDI-TOF mass spectrometry (MS) with peptide mass fingerprinting. For MS analysis, dried samples were rehydrated (2–3 µl of 0.3% trifluoroacetic acid, 1 mM ammonium citrate in 50:50 ACN:H2
O; plus an equal volume of saturated α-cyano-4-hydroxycinnamic acid matrix solution), spotted onto a target, and mass spectra obtained using an Applied Biosystems 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA). Spectra were calibrated via external standard and internal trypsin calibration. Mass spectra retrieved from MALDI-TOF MS were processed by GPS Explorer™ (ver. 3) MS data analysis software (Applied Biosystems, Foster City, CA) coupled with Mascot™ search engine (Matrix Science) for peak list generation and database search. Resulting peak lists were searched against the National Center for Biotechnology Information non-redundant (NCBInr) database, specifying “All Entries” or “Rattus
” species. See Supplementary Data S2
for annotated spectra, peak lists, and search criteria associated with top ranked hits. A positive protein identification for a given spot was accepted when a top ranked hit yielded a statistically significant probability-based MOWSE protein score and protein score confidence interval > 90% with a peptide count ≥ 6, protein coverage >20%, a predicted molecular weight that was relative to the protein spot position on the gel, and could be replicated across two or more separate 2-D DIGE gel experiments.
Fluorescence Imaging and Quantitative Image Analysis
For quantitative image analysis, 2-D DIGE gels were scanned for fluorescence imaging on a Typhoon 9400 laser scanner using ImageQuant software (GE Healthcare) at 100 µm resolution using photomultiplier tube (PMT) voltage settings below saturation for each dye (Cy3/5). Settings were determined for the first set of gels, both Cys- and Lys-CyDye labeled, then all further gels were scanned using the same PMT voltage settings, or ratio as necessary, to obtain non-saturation images. In-gel quantitative comparisons of fluorescence were completed using the Difference In-Gel Analysis (DIA) module of DeCyder Differential Analysis software (GE Healthcare). Fold change ratios, based on volume ratios of the individual spots and internally normalized by DeCyder, were determined and recorded for DeCyder-defined spots that corresponded to proteins previously identified by MS analysis. For each selected spot within a gel, the fold change was converted to percent DAQ-exposed mitochondrial protein fluorescence of control and averaged across all analyzed Cys-CyDye DIGE gels (n=6 total gels from 5 separate mitochondrial experiments) or Lys-CyDye DIGE gels (n=7 total gels from 5 separate mitochondrial experiments) using Excel (Microsoft Corp.). Images obtained from ImageQuant were prepared for presentation using Adobe Photoshop (Adobe).
PC12 Cell Culture and Mitochondrial Isolation
PC12 cells were maintained in DMEM supplemented with 7% horse serum (HS) and 7% fetal bovine serum (FBS). For differentiation, cells were subcultured on rat-tail collagen coated 100 mm plates at 1.5 × 106
cells/plate in DMEM supplemented with 1% HS, 1% FBS, and 0.1 µg/ml NGF for 6 days. Media was then replaced with fresh differentiating media with or without 150 µM DA and cells were incubated for 16 hrs. Cells were collected by force pipetting, rinsed with PBS, and isolated by centrifugation. Mitochondrial enriched fractions were prepared from 10 confluent plates in each group using methods similar to those for isolating rat brain mitochondria, with the modification of protease inhibitor cocktail (2 µl/ml) being present in the mitochondrial isolation buffer throughout the isolation process. Mitochondria were lysed in 2-D DIGE lysis buffer and final protein concentrations were determined by Bradford (1976
SDS-PAGE and Western Blot Immunodetection of Select Proteins
Lysed rat brain and PC12 cell mitochondrial protein samples (50 µg/lane) were run on 5–20% gradient SDS-PAGE (Hoefer ® Mighty Small II apparatus) and transferred to nitrocellulose (BioRad) for Western blot analysis via a BioRad Trans-Blot ® Semi-Dry Electrophoretic Transfer system. The membrane was removed, washed briefly in Tris-buffered saline (TBS), blocked in 0.2% w/v fat-free dry milk for 30min, rinsed briefly in TBS plus 0.1% Tween-20 (tTBS), and placed in a 1:1000 dilution of rabbit anti-MtCK or 1:5000 dilution of rabbit anti-mitofilin primary antibody in tTBS overnight at 4°C. Immunoreactive bands were visualized using the BioRad Immune-Star ® goat-anti-rabbit (dil 1:10,000) chemiluminescence detection kit and exposed to Biomax MR Film (Kodak). Mouse-anti-COXIV (dil 1:37,000; AbCam) and rabbit-anti-voltage-dependent anion channel 1 (VDAC1) (dil 1:2000; AbCam) were used as loading controls for rat brain mitochondria and for PC12 mitochondria, respectively. VDAC1 was selected because in parallel 2-D DIGE studies with PC12 cell mitochondria the protein did not significantly change following DA exposure (unpublished data). Films were digitally scanned and the densities of immunoreactive bands were determined using UN-SCAN-IT Gel (ver. 5.1) densitometry software (Silk Scientific; Orem, Utah).
Cys- and Lys-CyDye MS-identified proteins whose relative DAQ-exposed fluorescence values (as percent of control) fell outside of a defined range of 83.3–120% (±1.2 fold) were selected as different from control. The range represents two standard deviations in a Cy5-labeled control versus Cy3-labeled control gel analyzed by DeCyder (data not shown), and is the recommended threshold for determining significant change in DeCyder analysis. Statistical significance was determined using a 1-sample two-tailed Z-test on the DAQ-exposed mitochondrial protein spot volume intensities expressed as percent of control, as determined from DeCyder analysis. The Z-test is optimal, as DeCyder DIA software calculates changes between corresponding control and treated protein spots within a gel as a volume ratio of the two samples, generating one value of “fold change” for each protein spot that compares both groups. The ratios are then internally normalized across the entire constellation of labeled spots. Significance for each changed DA-exposed protein from control (valued at 100% control) was determined when p<0.01. The percent control values were directly calculated from the normalized DeCyder volume ratios. For Western blot analysis, rat brain mitochondria samples were run in duplicate or triplicate for each of n=6–7 separate experiments, and PC12 mitochondria samples were run in triplicate for each of n=4 separate experiments. Significance between group means was determined by two-factor ANOVA with replication followed by post-hoc Bonferroni tests.