3,4-Dihydroxyphenylethylamine, [8-14C] (14C-DA) was purchased from MP Biomedicals, Inc. (Irvine, CA). IEF DryStrips (7 cm 3-5.6pH, 4-7pH, and 6-11pH , and 18 cm 3-10pH), and CyDye™ DIGE Fluor Scarce Sample Labeling (Cysteine-reactive) and Minimal Labeling (Lysine-reactive) dye kits were purchased from GE Healthcare (Piscataway, NJ). Bradford Dye Reagent was purchased from BioRad (Hercules, CA). Promega Gold Mass Spectrometry Grade Modified Trypsin was purchased from Promega (Madison WI). Dopamine (DA), Protease inhibitor cocktail (cat#P2714), mushroom tyrosinase, retinoic acid, and most other chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) unless otherwise noted. The MtCK and mitofilin polyclonal antibodies used in this study were generated for our laboratory by Genemed Synthesis, Inc. (San Antonio, TX). Dulbecco’s modified Eagle medium (DMEM; Gibco) cell culture media, fetal bovine serum (FBS; HyClone), and 10,000 U/mL penicillin/10,000 μg/mL streptomycin (pen/strep; Gibco) were purchased from Invitrogen (Carlsbad, CA). All solutions were prepared using purified water from a Milli-Q system (Millipore Corp., Bedford, MA). Solutions for in-gel and on-blot trypsin digest procedures were prepared using HPLC-grade water from Fisher Biotech (Pittsburgh, PA), and HPLC-grade acetonitrile and spectrophotometric-grade methanol from Sigma-Aldrich (St. Louis, MO).
Mitochondrial Isolation and 14C-Dopamine Exposure Reactions
All animal procedures were approved by the Institutional 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 were isolated from adult male Sprague-Dawley (300-350g) rat brain tissue via differential centrifugation as previously described (Van Laar et al., 2008
). Mitochondrial pellets were resuspended in isolation buffer and kept on ice. Mitochondrial protein content was determined for the total suspension by the Bradford assay (Bradford, 1976
). Prior to experimental use, respiration measurements of the isolated mitochondria were made to ensure mitochondrial health, as previously described (Berman and Hastings, 1999
). A state 3/state 4 ratio above 6 was considered an indication of healthy, intact mitochondria.
Mitochondrial protein (4 μg/μL) was exposed to 150 μM DA or 14
C-DA (0.5-1 μCi; 150μM) and tyrosinase (300U/mL), to oxidize DA to DAQ, in reaction buffer (225 mM mannitol, 75 mM sucrose, 25 mM HEPES, and 1 mM EGTA, pH 7.4 with PIC) for 15 min at room temperature (RT). Mitochondria were then pelleted by centrifugation at 15,000 g for 15 min at 4°C. Control mitochondria underwent an identical procedure without DA present. Mitochondrial pellets 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) at 100 μL buffer/mg protein. Insoluble material was pelleted by centrifugation (16,000 × g for 1-2 min at RT) and discarded. Protein concentrations in the lysed control and DAQ-exposed samples were determined by the Bradford method (Bradford, 1976
SH-SY5Y Cell Culture and Dopamine Exposure
Proliferating SH-SY5Y cells were maintained in DMEM supplemented with 10% FBS and 1% pen/strep (SH media). For differentiation, cells were subcultured onto 6 cm or 6-well plates at 2 × 105 cells/mL. Culture media was exchanged for fresh SH media supplemented with 20 μM retinoic acid (SH differentiation media) 48 hr after plating, and every 48 hr thereafter for a total of 5 days of differentiation. On day 5, culture media was exchanged for fresh SH differentiation media supplemented with 150 μM 14C-DA (1 μCi/mL media) for 16 hr. Immediately following treatment, cells were collected by 1 min exposure to 0.25% trypsin with 2.21mM EDTA in HBSS (Mediatech; Herndon, VA) followed by force pipetting, rinsing with PBS, and centrifugation. The resulting cell pellet was rinsed with PBS, centrifuged, then lysed in DIGE lysis buffer supplemented with Chaps Cell Extract Buffer (Cell Signaling Technology; Danvers, MA) and PMSF.
2-D Gel Electrophoresis
For 2-D gel electrophoresis utilizing mini-gel SDS-PAGE, 100-250 μg total protein from 14C-DA exposed mitochondrial or SH-SY5Y cell lysate was loaded via sample cup on rehydrated 7cm pH 3-5.6, pH 4-7, or pH 6-11 DryStrips and isoelectrically focused on a Multiphor II electrophoresis unit according to manufacture’s instructions (GE Healthcare). Focused strips were equilibrated for 10 min at RT 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 strips were then subjected to electrophoresis on 12% SDS-PAGE gels utilizing a Hoefer Mighty Small II apparatus. Precision Blue markers (BioRad) were used as molecular weight standards.
Transblotting and Autoradiography of 14C Dopamine-modified Proteins
Following 14C-DA mini 2-D electrophoresis, proteins were transferred to PVDF membrane via a BioRad Trans-Blot Semi-Dry Electrophoretic Transfer system. For autoradiography, blots were air-dried and placed in Kodak autoradiogram transcreens with BioMax MS autoradiogram film (Kodak) in a film exposure cassette for 3 days to 3 weeks at -80°C.
Radiolabeled Protein Spot Excision and Trypsin Digest
Following autoradiography, PVDF blots were aligned with the autoradiograms. Using a sterile 2 mm tissue punch, small regions corresponding with spots on the autoradiogram were excised from the PVDF, and then subjected to trypsin digest by methods adapted from Bienvenut et al. (Bienvenut et al., 1999
). Briefly, spots were washed with 1:1 methanol and water, air-dried, and then submerged in 10μL 30% acetonitrile in 50 mM ammonium bicarbonate and 4μL 0.1mg/mL trypsin (Promega), and incubated 16-18 hr at RT with constant agitation. Following digestion, the supernatant was collected and saved in a separate tube. The membrane spots were then submerged in 20μL of 80% acetonitrile, and sonicated for 15 min at RT. The resulting supernatant was collected and added to the original digestion supernatant. The total supernatant was dried down by speed-vacuum without heating, and kept at 4°C under desiccation until MS analysis.
Cys- and Lys-CyDye labeling and 2-D DIGE
2-D DIGE analysis with either cysteine-reactive maleimide CyDye (Cys-CyDye) or lysine-reactive NHS-ester CyDye (Lys-CyDye) labeling of control and DAQ- or 14
C-DAQ-exposed mitochondrial protein was carried out as previously described (Van Laar et al., 2008
). Briefly, controls were paired with DAQ or 14
C-DAQ reacted samples from the same mitochondrial isolation, and experiments were completed for sample sets from multiple mitochondrial isolations. Cy5-labeled control and Cy3-labeled DAQ- or 14
C-DAQ-exposed samples were combined in equal protein amounts, generating the following DIGE gel experiments: (1) Cys CyDye Cy5 control vs. Cy3 DA, (2) Cys CyDye Cy5 control vs. Cy3 14
C-DA, (3) Lys CyDye Cy5 control vs. Cy3 DA, and (4) Lys CyDye Cy5 control vs. Cy3 14
C-DA. We previously determined through reciprocal labeling that there is no disparate or preferential labeling exhibited by the individual dyes (Van Laar et al., 2008
). Samples were isoelectrically focused on 18cm pH 3-10 DryStrips using a Multiphor II electrophoresis system (GE Healthcare). Focused DryStrips were equilibrated as described above and proteins were separated on 12% SDS-PAGE 1.5mm thick gels using a Hoefer SE600 Ruby Electrophoresis Unit.
Fluorescence Detection, Spot Picking, and In-gel Trypsin Digest
Immediately following the second dimension run, 2-D DIGE gels were scanned for Cy3 and Cy5 dye labeling using a Typhoon 9400 scanner with ImageQuant software (GE Healthcare) to obtain a 100-200 μm resolution image of the gel. Immediately following imaging, 14
C-DA 2-D DIGE gels were transblotted to PVDF or nitrocellulose membranes. Non-radioactive 2-D DIGE gels were fixed overnight in 40% MeOH, 1% acetic acid solution at 4°C. Spots of interest were excised using an automated spot picker, designed by Dr. Jonathan Minden of Carnegie Mellon University (instrumentation housed in the University of Pittsburgh Genomics and Proteomics Core Laboratories). Immediately following excision from 2-D DIGE gels, gel plugs were washed and carried through a previously described in-gel trypsin digest procedure (Van Laar et al., 2008
C-DA 2-D DIGE experiments, transblots were subjected to autoradiography. Digital scan images of the autoradiogram and the corresponding DIGE gel were digitally merged and compared visually. Images were aligned based on recognizable landmarks. Fluorescence-labeled protein spots that aligned with radioactive spots were considered spots of interest. For correlating protein spots that we did not already have an identification for based on previous data (Van Laar et al., 2008
), corresponding spots on non-radioactive DIGE gels were picked for identification, as described above.
MS and MS/MS Analyses for Protein Identification
For MS and MS/MS analyses, dried trypsin-digested samples were rehydrated in 2-3 μl of 0.3% trifluoroacetic acid, 1 mM ammonium citrate in 50:50 acetonitrile/water, and an equal volume of saturated α-cyano-4-hydroxycinnamic acid matrix solution, and then spotted onto a MALDI target plate at 42°C. MS and MS/MS spectra were obtained using an Applied Biosystems 4700 MALDI-TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA) and processed by GPS Explorer™ (ver. 3) data analysis software (Applied Biosystems) coupled with Mascot™ search engine (Matrix Science) for peak list generation, database search, and statistical analyses. MS and/or combined MS and MS/MS spectra were searched against the National Center for Biotechnology Information non-redundant (NCBInr) and SwissProt databases, specifying “Rodentia” or “Rattus” species for rat brain mitochondria samples, and “Homo sapiens” species for SH-SY5Y samples. Trypsin digest was specified for searches, and MS and MS/MS peak filtering were set at a minimum S/N ratio of 10, with a peak density filter of 50 and maximum 65 peaks allowed. Allowed mass ranges were 800-4000 Da for MS peaks and 20-60 Da under precursor for MS/MS. Precursor tolerance was set at 50 to 100 ppm and MS/MS fragment tolerance was set at 0.2 to 0.4 Da, allowing 1 missed cleavage. Modifications specified included fixed or variable cysteine carbamidomethylation and methionine oxidation.
For peptide mass fingerprinting, a positive protein identification was accepted when a top ranked hit yielded: (1) a probability-based molecular weight search (MOWSE) protein score confidence interval percentage (C.I.%) > 95%, (2) peptides matched ≥ 6, (3) a predicted molecular weight that was appropriate given the migration of the protein spot on the gel or blot, and (4) MS identification for a given 14C-DA-labeled spot could be replicated across two or more separate experiments. Five statistically significant protein identities are also noted that were obtained via MS peptide mass fingerprinting analysis in only one 14C-DA exposure experiment, though corresponding 14C-DA conjugated spots were visible in blots from multiple experiments. These identities are noted in Tables and . Identification for a particular spot was accepted for combined MS and MS/MS (MS + MS/MS) results that yielded (1) a top ranked hit with both MS probability-based MOWSE protein score and MS/MS spectra total ion score C.I.% each > 95%, (2) total peptides matched ≥ 6 and/or significant ion score for ≥ 2 unique peptides, (3) a predicted molecular weight that was appropriate given the migration of the protein spot on the blot, and (4) a corresponding 14C-DA-labeled spot was present in blots from multiple experiments. Total ion scores were calculated from weighted ion scores for individual peptides that were matched to a given spot identity. C.I.% values are derived from the probability-based MOWSE scores; values >95% suggest identities are significant, and not random matches. For each protein identity provided, the next non-homologous protein identified using Mascot did not meet our guidelines for acceptability as described above, suggesting that every identified protein was uniquely assigned to its corresponding spot (data not shown).
Identified DA-Conjugated Proteins from DAQ-Exposed Rat Brain Mitochondrial Fractions
Identified DA-Conjugated Proteins from DA-Exposed SH-SY5Y Cells
Western Blot Detection of Mitofilin & MtCK
Following autoradiography, the transblots generated from 14C-DA 2-D DIGE gels were carried through the Western blot detection procedure. The membrane was washed and placed in a 1:1000 dilution of rabbit anti-MtCK or 1:5000 dilution of rabbit anti-mitofilin primary antibody 16-18 hr at 4°C. Membranes were developed using the BioRad Immune-Star® goat-anti-rabbit Chemiluminescence Detection kit, exposed to Biomax MR film (Kodak), and developed for imaging. Using recognizable landmarks for alignment, scanned images of the Western blot and the autoradiogram were digitally merged with the ImageQuant scan of the fluorescent DIGE gel for visual comparison.