Subjects and tissue
Four adult male rhesus monkeys (Macaca mulatta) were restrained with Telazol, given intravenous heparin and then an overdose of intravenous sodium pentobarbital. After the confirmed absence of brain stem reflexes was established, the monkeys were transcardially perfuse with phosphate buffered saline to evacuate brain vasculature. Brains were blocked using a rhesus monkey brain matrix that allows 4 mm coronal blocks at various AP locations (Electron Microscopy Sciences, Ft. Washington, PA). Blocks were divided into their two component hemispheres – one for fresh frozen sections at -80°C and the other for dissection of blocks for paraffin embedding. Brain tissue from these monkeys was frozen within 40 minutes of the time of death. All experiments were conducted in accordance with the National Institutes for Health Guide for the Care and Use of Laboratory Animals.
Protein isolation and fractionation
One hundred and fifty milligram punches were dissected from NAc from each subject. A steel mortar and pestle chilled in dry ice were used to pulverize the frozen brain tissue from each subject separately into a dry homogenate in the presence of liquid nitrogen. Tissue proteins from each subject were fractionated into membrane, nuclear and cytosolic fractions as described previously [25,26]. The tissues were homogenized in 10 mM HEPES, 10 mM NaCl, 1 mM KH2PO4, 5 mM NaHCO3, 1 mM CaCl2, 0.5 mM MgCl2, 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 10 mM benzamidine, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 1 μg/ml pepstatin. The tissue homogenate from each subject was centrifuged using a swinging bucket rotor (Beckman Coulter SW55Ti) at 5333 × g for 5 min. The supernatant from each subject (cytosolic and crude membrane fraction) was further centrifuged at 59,255 × g for 30 min at 4°C and the cytosolic supernatant was stored at -80°C.
SDS-Polyacrylamide Gel Electrophoresis (PAGE)
The protein quantitation for SDS-PAGE was accomplished using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL). Laemmli sample buffer was used to achieve equivalent protein concentrations for all samples. Thirty micrograms of protein from each sample was heated to 95°C for 5 min and electrophoresed on 10% Tris-HCl SDS-PAGE gels (BioRad). Gel fixation, staining by Pro-Q® Diamond phospho-protein stain and the SyproRuby™ staining of the SDS-PAGE was completed as described in detail for 2D-PAGE.
Extraction of proteins for 2D-PAGE
The cytosolic protein fraction from each subject was precipitated by 2-D clean-up kit (GE Healthcare) at 20°C overnight, pelleted by centrifugation (12,000 × g for 5 min) then air-dried for 2 min. Next, pellets were dissolved in re-hydration buffer [RB; 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate. The protein solution in RB was supplemented with immobilized pH gradient buffer 2% and 2% dithiothreitol (DTT) (10 mg/ml). The protein concentrations were established by the urea and detergent compatible 2D-Quant kit (Amersham Biosciences).
2D-PAGE
Two hundred microgram aliquots of cytosolic proteins from each subject were diluted in 400 μl of RB and increased to a final volume of 450 μl with destreak reagent (GE Healthcare). Immobiline™ DryStrips (240 × 3 × 0.5 mm, pH 4–7 linear) were re-hydrated for 10 hr on an Amersham Pharmacia Biotech IPGphor [27] followed by sequential isoelectric focusing (IEF) of samples as follows: 100 V for 100 V-hr, at 500 V for 500 V-hr, at 1000 V for 1000 V-hr, at 4000 V for 4000 V-hr, at 8000 V for 13500 V-hr (gradient) and at 8000 V for 60,000 V-hr. Fifty μA current was passed per strip maintaining the platform temperature at 20°C. Next, strips were equilibrated by adding a solution containing; 6 M urea, 1.5 M Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS and 2% (w/v) DTT and gentle rocking for 10 min, to reduce the disulfide bonds. Next, a solution containing 6 M urea, 1.5 M Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS and 2.5% (w/v) iodoacetamide was added to block sulphydryl groups.
After equilibration, the IPG strips were transferred to 2-D slab gels using 0.6% agarose stacking gel. The proteins were further separated on the basis of their molecular weight on 12.5% SDS-PAGE (2400 × 2000 × 1 mm) at a constant 4 W per gel until the dye front reached the end of the gel using the Ettan Dalt II System (GE Healthcare, Piscataway, NJ). Ten μl of the molecular mass marker (Amersham Rainbow marker RPN 800) was loaded on 2 mm2 filter paper which was placed on the basic end of the IPG strip [28,29]. To visualize the proteome from this specific pH and mass range, gels were stained with Sypro Ruby™ stain overnight. The excess stain was removed by 10% methanol, 6% glacial acetic acid for 20 minutes. Protein spots of interest were subjected to MALDI-TOF-TOF [30].
In-gel trypsin digestion
Individual protein spots from the 2-D gels were excised with 1.5 mm diameter gel cutter (The Gel Company, San Francisco, CA) [31]. The gel spots were washed for 20 min, twice in 100 μl of solution of 50 mM ammonium bicarbonate, 50% methanol (v/v) in distilled water and once in 75% acetonitrile in distilled water for 30 min or until the gel plugs turned opaque. Twenty micrograms of lyophilized trypsin (883 pmol; Promega, Madison, WI) was reconstituted in 1 ml of 20 mM ammonium bicarbonate and incubated for 15 min at 37°C. The gel fragments were dried by vacuum centrifugation and then incubated overnight with 10 μl (200 ng) of trypsin at 37°C. The supernatant from trypsin digest was transferred to a low retention 96-well plate. Peptides from the gel pieces were sequentially extracted twice in 100 μl of extraction buffer [50% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid (TFA) in distilled water (DW)]. The original tryptic supernatant and the supernatants from two sequential extractions were combined and dried in a vacuum centrifuge. The dried peptides from each gel plug were dissolved in 5 μl of 50% (v/v) acetonitrile, 0.1% trifluoroacetic acid in distilled water and 0.5 μl deposited on the stainless-steel MALDI target plate. After drying, the spot residue was mixed with 0.5 μl of 5 mg/ml of α-cyano-4-hydroxy-cinnamic acid (CHCA; Sigma-Aldrich, St. Louis, MO) in 50% (v/v) acetonitrile, 0.1% trifluoroacetic acid in distilled water[32].
MALDI-TOF-TOF
Mass spectrometry analyses were performed using the Applied Biosystems 4700 Proteomics Analyzer (MALDI-TOF-TOF; Foster City, CA) in reflector mode for positive ion detection. The laser wavelength and the repetition rate were 355 nm and 200 Hz, respectively. All the MS spectra resulted from accumulation of at least 2000 laser shots. The peak detection criteria used were; minimum S/N of 8, local noise window width mass/charge (m/z) of 200 and minimum full-width half-maximum (bins) of 2.9. The mass spectra were calibrated using the three trypsin auto digest products: fragment 100–107 ([M + H]+ = 842.51 Da), fragment 90–99 ([M + H]+ = 1045.556 Da) and fragment 50–69 ([M + H]+ = 2211.105 Da) [33]. A maximum of the ten strongest precursor ions per sample were chosen for tandem mass spectrometry (MS/MS) analysis. The following monoisotopic precursor selection were used for the MS/MS: minimum S/N filter of 10, excluding the most commonly observed peptide peaks for trypsin and keratin, and excluding the precursors within 150 resolution. In the TOF1 stage, all ions were accelerated to 1 kV under conditions promoting metastable fragmentation. The peak detection criteria used were; S/N of 8 and local noise window width of 250 (m/z).
De novo sequencing
The PEAKS Studio 4.0 (Bioinformatics Solutions, Waterloo, Ontario, Canada) de novo sequencing software was used for automated de novo sequencing followed by manual confirmation of most sequences generated. A parent- and fragment-mass error tolerance of 0.08 u; trypsin as the protease with one maximum missed cleavage allowed; deconvolute the charge state in the spectra to generate a spectra in which each monoisotopic peak becomes singly charged; partial modification of cysteine (carbamidomethyl-cysteine) and methionine (oxidized), were used as the de novo sequencing parameters. The most abundant peptide fragments 'b-ions and y-ions'; the less abundant peptide fragments 'a-ions'; the neutral losses of water and ammonia for b-ions and y-ions; as well as the immonium ions were utilized to develop confident and complete peptide sequences de novo from MS/MS spectra [24]. Since the Macaca mulatta has an incomplete protein database, the sequences generated from each spectrum were used for protein identification by sequence homology in the mammalian database using either the PEAKS or SPIDER software (Software Protein Identifier). Therefore, the SPIDER software was useful when the de novo sequencing gave partially correct sequence tags and at instances where a segment of amino acids was replaced by another segment with approximately same masses [Han, Y JBCB; (3) 2005; 697–716]. The algorithm used for determining the probability based scoring with a given mass spectrum is described in detail by Ma B et al [24]. Protein identification was confirmed by checking the protein mass and pI accuracy [30].
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