Proteomic analysis of the retinal rod outer segment disks

Proteomic analysis of the retinal rod outer segment disks
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  See discussions, stats, and author profiles for this publication at: Proteomic Analysis of the Retinal Rod OuterSegment Disks  Article   in  Journal of Proteome Research · August 2008 DOI: 10.1021/pr7006939 · Source: PubMed CITATIONS 45 READS 47 13 authors , including: Some of the authors of this publication are also working on these related projects: Public Dissemination of Science   View projectI'm working on true myelin role. You can see our last paper Support of Nerve Conduction byRespiring Myelin Sheath: Role of Connexons. 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All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Proteomic Analysis of the Retinal Rod Outer Segment Disks Isabella Panfoli,* ,†,# Luca Musante, ‡,§,#  Angela Bachi,* , | Silvia Ravera, † Daniela Calzia, †  Angela Cattaneo, | Maurizio Bruschi, ‡,§ Paolo Bianchini, ⊥  Alberto Diaspro, ⊥ , ∇  Alessandro Morelli, † Isidoro M. Pepe, † Carlo Tacchetti, O and Giovanni Candiano ‡ Department of Biology, University of Genoa, V.le Benedetto XV 3, 16132 Genova, Italy, Laboratory on Pathophysiology of Uraemia, L.go G. Gaslini 5, 16147 Genova, Italy, Renal Child Foundation, L.go G. Gaslini, 5 16147 Genova, Italy, DIBIT-San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milano, Italy, Laboratory  for Advanced Microscopy, Bioimaging, and Spectroscopy (LAMBS)-MicroSCoBiO Research Center, Department of Physics, University of Genoa, Via Dodecaneso 33, 16146 Genova, Italy, IFOM  s The FIRC Institute for Molecular Oncology, Via Adamello 16, 20139 Milan, Italy, and IFOM Center for Cell Oncology and Ultrastructure, Department of Experimental Medicine, University of Genoa, Via de Toni 14, 16132, Genoa, Italy  Received October 26, 2007 The initial events of vision at low light take place in vertebrate retinal rods. The rod outer segmentconsists of a stack of flattened disks surrounded by the plasma membrane. A list of the proteins thatreside in disks has not been achieved yet. We present the first comprehensive proteomic analysis of purified rod disks, obtained by combining the results of two-dimensional gel electrophoresis separationof disk proteins to MALDI-TOF or nLC-ESI-MS/MS mass spectrometry techniques. Intact disks wereisolated from bovine retinal rod outer segments by a method that minimizes contamination from innersegment. Out of a total of 187 excised spots, 148 proteins were unambiguously identified. An additionalset of 61 proteins (partially overlapping with the previous ones) was generated by one-dimensional(1D) gel nLC-ESI-MS/MS method. Proteins involved in vision as well as in aerobic metabolism werefound, among which are the five complexes of oxidative phosphorylation. Results from biochemical,Western blot, and confocal laser scanning microscopy immunochemistry experiments suggest thatF 1 F o -ATP synthase is located and catalytically active in ROS disk membranes. This study represents astep toward a global physiological characterization of the disk proteome and provides informationnecessary for future studies on energy supply for phototransduction. Keywords:  disks  •   F 1 F o -ATP synthase  •   confocal laser scanning microscopy  •   MALDI-TOF  •   nLC-ESI-MS/MS  •   redox chain complexes  •   Rod Outer Segment  •   two-dimensional polyacrylamide gelelectrophoresis Introduction The rod cell consists of an inner and an outer segment. TheRod Inner Segment (RIS) contains the organelles required forthe metabolic processes. The Outer Segment (ROS) is a stack of floating sealed flattened membrane vesicles, the disks, thatare surrounded by the plasma membrane. Disks, where lightis captured by the photopigment rhodopsin (Rh), are the siteof the initial events of the visual transduction cascade. 1,2 ROSconstantly undergo renewal 3,4 by post-transductional  in situ  modifications: 5 new disks formed at the cilium of the outersegment from evaginations of the plasma membrane areprogressively displaced toward the apical tip, and finally shedby the retinal pigment epithelium (RPE). 4,6 In vertebrates, thetransit of disks from the base to the tip of the ROS requiresapproximately 10 days. 4 This localization is correlated to adecrease in membrane cholesterol content in parallel with theacquisition of functionality. 5 Disk membrane age is coincident with spatial location within the ROS.Comprehensive understanding of as yet unanswered ques-tions about visual phototransduction, such as GTP/ATP supply in ROS and light adaptation, would require complete identi-fication of the proteome of disk membranes, where most of the major players in photoreceptor-specific functions arepresent. As yet, no proteomic studies have been conducted on disks. A separation of ROS proteins was previously conducted by afirst-dimensional pH 6 - 8 ampholine focusing and a second-dimensional SDS-PAGE. 7 More recently, in a two-dimensionalpolyacrylamide gel electrophoresis (2D-PAGE) study on thephotoreceptor cell layer of the bovine retina consisting mostly  * Corresponding author: Dr. Isabella Panfoli, Universita` di Genova,Dipartimento di Biologia V.le Benedetto XV,3, 16132 Genova, Italy. Phone, ++ 39 010 353.7397; fax, ++ 39 010 353.8153, e-mail, author for proteomic analysis: Dr. Angela Bachi, DIBIT, SanRaffaele Scientific Institute, Via Olgettina, 58, I-20132 Milano, Italy. Phone, + 390226434927; fax,  + 390226434153; e-mail, † Department of Biology, University of Genoa. ‡ Laboratory on Pathophysiology of Uraemia. § Renal Child Foundation. # These authors contributed equally to this work. | DIBIT-San Raffaele Scientific Institute. ⊥ Department of Physics, University of Genoa. ∇ IFOM s The FIRC Institute for Molecular Oncology. O Department of Experimental Medicine, University of Genoa. 2654 Journal of Proteome Research  2008, 7, 2654–2669   10.1021/pr7006939 CCC: $40.75  ©  2008 American Chemical SocietyPublished on Web 05/20/2008  of intact rods, only three proteins were identified by massspectrometry (MS). 8 Proteomic 2D-PAGE studies were con-ducted on the porcine interphotoreceptor matrix  9 and on thehuman Retinal Pigmented Epithelium (RPE). 10 The study presented here was set up to identify ROS disk proteome. Disks were isolated by the procedure of Smith andLitman, 11 that yields a pure preparation, with limited contami-nation by RIS proteins. Matrix-assisted laser desorption time-of-flight (MALDI-TOF) mass spectrometry (MS) or nano liquidchromatography-electrospray ionization MS (nLC-ESI-MS/MS)techniques were applied, in combination with 2D-PAGE sepa-ration of disk proteins. An additional data set was generated with a limited number of bands between 70 and 200 kDa,excised from a one-dimensional (1D-PAGE) separation of disk proteins, analyzed by nLC-ESI-MS/MS. Experimental Section Preparation of Bovine ROS.  Intact ROS were prepared 12 under dim red light from 20 retinas of bovine eyes (obtainedfrom a local slaughterhouse), at 4  ° C. Briefly, 5 retinas wereadded to 5 mL of Mammalian Ringer (MR) consisting of (inmM): 157 NaCl, 5 KCl, 7 Na 2 HPO 4 , 8 Na 2 HPO 4 , 0.5 MgCl 2 , 2CaCl 2 , pH 6.9, and 35% sucrose (w/v), in the presence of protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO) andampicillin (100  µ g/mL). Homogenization of bovine retinas, astandard procedure for purification of bovine outer segments, was avoided, as it is a harsh treatment. Short pulse vortexing (for 15 s for 2 times at 300 rpm) was employed, so that theretinal shake is gentle enough not to cause major disruptionof other retinal cell types. The retinal suspension was centri-fuged at 6000 g   for 10 min at 4  ° C (SS-34 rotor; Sorvall KendroLaboratory Products, Newtown, CT). To maximize recovery, theprocedure was repeated twice on each set of 5 retinas. The pink supernatants representing the ROS suspension were filteredthrough cheesecloth, pooled and carefully diluted to 10%sucrose (w/v) with MR. Suspension was then centrifuged inthe same SS-34 rotor at 4000 g   for 40 min. ROS pellets wereretained. Light microscopic observation of ROS from thesucrose gradient from each preparation was routinely performed. Osmotically Intact Disk Preparation.  Osmotically intactdisks were obtained from ROS following the procedure of Smithand Litman, 11 as previously reported. 13 Briefly, ROS wereallowed to burst for 3 h in distilled water, 70  µ g/mL leupeptinand 100  µ g/mL ampicillin, then the suspension was layered with cold water over a solution of distilled water with 5% (w/v) Ficoll (Sigma-Aldrich, St. Louis, MO) and centrifuged for 2 hat 25 000 rpm in a Beckman FW-27 rotor. Disks were collectedas a pink layer at the interface between water and 5% Ficoll. Rhodopsin Determination.  Rh concentration was deter-mined spectrophotometrically (molar extinction coefficient of 41 000 cm - 1 · M - 1 ) 14 by measuring the difference in 500 nmabsorption between spectra recorded before and after exhaus-tive bleaching (green light, for 5 min). Absorbance spectrumof disk suspensions (0.06 mg/mL, in distilled water) wasrecorded with a dual-beam spectrometer (UNICAM UV2, Analytical S.n.c., Italy). Rhodopsin concentration in the srcinalpreparation of disks was 0.8 mM. Transmission Electron Microscopy (TEM) of Osmotically Intact Disk Preparations.  For TEM analysis, disks (1.2 mg totalprotein) were fixed in 2.5% (v/v) glutaraldehyde in 0.1 Mcacodylate buffer, pH 7.4, for 10 min. Sample was then washed with 0.1 M cacodylate buffer, pH 7.4, then 2% (w/v) osmiumtetroxide in 0.1 M cacodylate buffer (pH 7.4) was added to thesample. After 10 min incubation, osmium solution was dis-carded and samples were treated with a 1% (v/v) aqueoussolution of uranyl acetate, then dehydrated through a gradedethanol series. Samples were embedded in EPON resin andpolymerized overnight at 42  ° C, followed by 48 h at 70  ° C. Ultrathin sections were obtained using a Leica Ultracut E microtomeand analyzed with a Tecnai 12 G2 (FE1, Eindhoven, TheNetherlands) electron microscope. Immunohystochemical Preparations for Confocal LaserScanning Microscopy (CLSM).  For CLSM, disks (60  µ g of totalprotein) were subjected to 15 passages through a needle (25gauge) to eliminate aggregates. Treatments were conducted inEppendorf vials (50  µ L final volume), as previously described 13  with primary Ab’s: 2  µ g/mL of mouse monoclonal antibody (Ab)against bovine Rh (C-terminus, last 9 amino acids, ChemiconInt., Temecula, CA, Cat No. MAB5356); 2  µ g/mL mouse mono-clonal Ab against rabbit Na/K-ATPase R  1 (C464.6) (Santa CruzBiotecnhnology Inc., Santa Cruz, CA); or 2  µ g/mL and rabbitpolyclonal Ab anti- R  /    subunits of human recombinant F o F 1 - ATP synthase (a kind gift of Prof. Zanotti, University of Bari,Italy  15 ) and secondary Ab’s (anti-mouse or anti-rabbit IgGconjugated with Cy3 or Cy5 fluorochromes (Molecular Probes,Invitrogen Corporation, Carlsbad, CA), diluted in 10 mMPhosphate Buffered Saline (PBS). In controls, disks were treated with secondary Ab’s only. Incubation of disks with secondary  Ab’s yielded negligible immunoreactivity, confirming the speci-ficity of disk-surface interaction of the Ab with the proteintargets (data not shown). CLSM Imaging.  CLSM imaging was conducted on the abovesamples at 23  ° C. Image acquisition was performed using aLeica TCS SP2 AOBS CLSM, equipped with a 405-nm 10-mW laser diode, with an internal spectrophotometer function.Images were collected using a 100 ×  oil N.A. 1/4 1.4 objectiveHCX PL APO (Leica Microsystems GmbH, Wetzlar, Germany).Imaging of the sample was conducted using the 514-nm and647-nm lines of a 20-mW Argon ion laser. To detect thepresence/absence of the dyes (Cy3; Cy5) images were analyzedto determine the related mean intensity values. A spectralevaluation was made possible by the spectral characteristicsof the Leica SP2 AOBS scanning head. The spectral window used for collecting fluorescence was chosen in accordance tothe reported emission spectra. 16 Fluorescence of Cy3 and of Cy5 was excited with a beam of wavelength at 543.5 and 647nm, respectively. Look-up table liner was used to collect allthe acquisitions. The spectral window used for collecting fluorescence was 560 - 660 nm for Cy3 and conjugates wereexcited maximally at 650 nm and fluoresced maximally at660 - 780 nm for Cy5. The resulting images were acquired,stored, processed and visualized with the Leica ConfocalSoftware (LCS). Two-Dimensional Gel Electrophoresis (2D-PAGE).1. Sample Preparative Analysis.  Disks were treated according to Herbert et al., 17  with minor modifications. Briefly, 2 mg of disk proteins were lyophilized and then solubilized in 100  µ Lof a denaturing/reducing solution, containing (final concentra-tions): 7 M urea, 2 M thiourea, 65 mM 1,4-dithioerythritol(DTE), 40 mM Tris, and 0.1 mM ethylenediaminetetraaceticacid (EDTA). After 1.5 h, 4 M ratio of iodacetamide (IAA, 260mM final) was added, and the sample was incubated for 1 h atroom temperature. Excess IAA was eliminated by adding anequimolar amount of DTE, to prevent overalkylation. Then, thesample was delipidated and desalted by precipitation in tri n  -butylphosphate/acetone/methanol (1:12:1). Two milliliters of  Proteomics of Retinal Rod Disks   research articles Journal of Proteome Research  •  Vol. 7, No. 7, 2008  2655  this mixture were added to the sample and incubated end-over-end with agitation at 4  ° C for 90 min. The precipitate wascollected by centrifugation at 2800 g   for 20 min at 4  ° C. Thepellet was washed with the same solution, then centrifuged andair-dried. 18 The pellet was finally dissolved in the focusing solution, that is, 7 M urea, 2 M thiourea, 4% (w/v) 3-[(3-cholamidopropyl) dimethylammonium]-1-propane sulfonate(CHAPS) and 10 mM Tris. 19 2. Rehydration of Immobilized pH Gradient (IPG) GelStrips.  Homemade “soft IPG” 19 (24 cm) nonlinear pH 3 - 10Immobiline dry strips (IPG) were rehydrated 20 in a solutioncontaining (final concentrations): 7 M urea, 2 M thiourea, 4%(w/v) CHAPS, 1.6% (w/v) carrier ampholytes with a wide-rangemixture (40% pH 4 - 8, 60% pH 3.5 - 10) and traces of Bro-mophenol Blue. Reswelling was carried out overnight at roomtemperature. 3. Immobiline Dry Strips (IPG, First Dimension).  Allsamples were routinely loaded by cup loading, at the basic endof the rehydrated IPG strips, and covered with low-viscosity paraffin oil. Isoelectric focusing was performed at 17  ° C. Thestrips were run using the following voltage gradient program:start at 100 V for 2 h, step and hold; 350 V for 1 h, step andhold; 450 V for 1 h, step and hold; 600 V for 1 h, step and hold;600 - 5000 V linear gradient for 5 h; 5000 V for 10 h, step andhold; 5000 - 8000 V linear gradient for 2 h; 8000 V for 2 h, stepand hold, for a total of 120 kVh and 100  µ  A (total time, 24 h).Before the second-dimension run, preparative IPG strips wereequilibrated in the strip tray with a solution of 0.05 M Tris-HCl buffer (pH 6.8) containing 6 M urea, 30% (w/v) glyceroland 5% (w/v) sodium dodecyl sulfate (SDS) for 20 min. 4. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electro-phoresis(SDS-PAGE)(SecondDimension). The vertical seconddimension was performed on 220  ×  260  ×  1.5 mm slabs of polyacrylamide-piperazine diacrylamide gradient gels (T 8 - 16%;C 2.6%) in an Anderson IsoDALT electrophoresis system(Hoefer Scientific Instruments). Stacking gels were not em-ployed. IPG strips were put on top of the slab gels and sealed with the upper buffer solution containing 0.5% (w/v) agarose.The gels were run at 12  ° C at 100 mA/gel constant current for1 h, then current was decreased to 60mA/gel and run continuedovernight, for a total of 18 h. After separation, proteins werefixed in 40% (v/v) ethanol and 10% (v/v) acetic acid for 1 hand gels were stored in 5 (v/v) ethanol and 5 (v/v) acetic acid,followed by silver staining  21 for the analytical image. “Bluesilver” colloidal Coomassie was performed for preparative massspectrometry analysis. 22 Stained gels were digitized by using aGS800 photometer and analyzed with the PDQuest software(Bio-Rad). 5. Analytical 2D-PAGE for Rhodopsin (Rh) Identification. To identify Rh, 50  µ g of lyophilized disk proteins were treatedas for preparative analysis. IPG were homemade with a pHrange of 2.5 - 7. The sample was loaded by cup loading at thebasic end of IPG. Isoelectric Focusing (IEF) in Cylindrical Gels.  IEF in cylin-drical gels (20 mm length, 1 mm diameter) was performedaccording to Anderson and Anderson 23  with minor modifica-tions. The Ampholyte content was (20 mL/L) 2.5 - 4.0, 3.5 - 10and 9.0 - 11 (1:9:1 by vol.). Fifty micrograms of total disk proteins were loaded. Run was conducted at constant voltage with 800 V per gel, for 14 h. The second dimension was carriedout as described above for IPG strips. Protein Identification by MALDI-TOF MS Analysis of 2DGels.  Spots of interest were manually excised from gels,reduced, alkylated and digested overnight with bovine trypsin(Roche Diagnostics Corp., Indianapolis, IN). 24 One microliterof the supernatant was used for MS analysis on a MALDI-TOF Voyager-DE STR (Applied Biosystems, Foster City, CA) massspectrometer. Spectra were accumulated for a mass range of 750 - 4000 Da with a mean resolution of about 15 000.Spectra were internally calibrated using trypsin autolysisproducts and processed  via   Data Explorer software version4.0.0.0 (Applied Biosystems). Alkylation of cysteine by carba-midomethylation and oxidation of methionine were consideredas fixed and variable modifications, respectively. Two missedcleavages per peptide were allowed, and an initial masstolerance of 50 ppm was used in all searches. Peptides withmasses correspondent to those of trypsin and matrix wereexcluded from the peak list. Proteins were identified by searching against a comprehensive nonredundant proteindatabase using both Pro-Found and MASCOT programs via theInternet. 25  When several isoforms were retrieved, we manually searched for unique peptides, when present, to discriminatebetween the different isoforms. ProteinIdentificationbynLC-ESI-MS/MSof2DGels.  A totalof 5 - 8  µ L of trypsin digested sample was injected in a capillary chromatographic system Agilent 1100 Series equipped with aNano Pump, Iso Pump, Degaser and a 8  µ L injection loop(Agilent, Germany). Peptide separations occurred on a RP nanocolumn (75  µ m i.d.  ×  15 cm), packed with C18 PepMap100material (LC Packings), or on a 10 - 15 cm fused silica emitter(75  µ m inner diameter, 360  µ m outer diameter; ProxeonBiosystems Odense, Denmark) used as analytical column. Theemitter was packed in-house with a methanol slurry of reverse-phase, fully end-capped with Reprosil-Pur C18 3  µ m resin (Dr.Maisch, GmbH, Ammerbuch-Entringen, Germany) using apressurized “packing bomb” operated at 50 - 60 bar.Mobile phases consisted of water with 2% (v/v) acetonitrileand 0.2% formic acid (v/v) (buffer A) and acetonitrile with 2%(v/v) water and 0.2% formic acid (v/v) (buffer B). A 60-mingradient from 10 to 80% buffer B at a constant flow rate of 200nL/min was used for peptides separation. Eluting peptides wereionized through a Pico Tip FS360 - 20 - 10 (New Objective)supported by a nanoelectrospray ion source (Proxeon Biosys-tems, Odense, Denmark) and analyzed on an API QStarPULSAR (PE-Sciex, Canada) mass spectrometer. Analyses wereperformed in positive ion mode; the HV Potential was set uparound 1.8 - 2.0 kV. Full scan mass spectra ranging from  m  / z  350 to 1350 Da were collected, and for each MS spectrum, thetwo most intense doubly and triply charged ions peaks in therange from 350 to 1350 were selected for fragmentation. MS/MS spectra data files from each chromatographic run werecombined and converted to mgf files using Mascot.dll (version1.6.0.21) through Analyst QS 1.1 (Applied Biosystems) andsearched against mammalian NCBInr (download  20 070 216 ;4 626 804 sequences; 1 596 079 197 residues ) with Mascotsearch engine (version 2.1.04, Matrix Science, London, U.K.).Mass tolerance was set to 200 ppm and 0.3 Da for precursorand fragment ions, respectively. Searches were performed withtrypsin specificity, alkylation of cysteine by carbamidomethy-lation, and oxidation of methionine as fixed and variablemodifications, respectively. Criteria used to evaluate the quality  research articles  Panfoli et al. 2656 Journal of Proteome Research  •  Vol. 7, No. 7, 2008  of identifications for MS/MS data were a protein Mascot scoregreater than 100 with at least 2 peptides fragmented perprotein. One-Dimensional Denaturing Gel Electrophoresis (1D-PAGE).  Denaturing 1D-PAGE was performed according toLaemmli, 26 using either a Mini Protean III (Bio-Rad, Hercules,CA) (60  ×  80  ×  1.5 mm), or a PROTEAN II xi | XL VerticalElectrophoresis Cells (Bio-Rad, Hercules, CA) apparatus (200 ×  200  ×  0.75 mm). Separating gel was a gradient from 8% to14% (w/v) SDS-PAGE. Run was conducted at 4  ° C, at 70 mA/gel. For run in PROTEAN II xi | XL, disk proteins wereprecipitated by addition of 6% (w/v) trichloroacetic acid (TCA)and 10 mM Na 2 HPO 4,  chilled on ice for 1 min, and thencentrifuged at 14 000 rpm for 2 min in Eppendorf Centrifuge,supernatant was discarded, and pellets were rapidly washedtwice with 80  µ L of ethyl ether, by centrifugation. Final pellets were air-dried and resuspended using 8% SDS (w/v) in 125 mMTris-HCl (pH 6.8) and 1.25% (v/v) DTT and incubated for 15min. Then, the samples were boiled for 5 min, and a secondsolution containing 40% (w/v) sucrose and 0.008% (w/v)Bromophenol blue was added.Protein Molecular Weight (M.W.) markers were purchasedfrom Fermentas (Fermentas Life Sciences, Hanover, MD, Catno. SM#1811), or from Sigma (Sigma-Aldrich, St. Louis, MO;Cat no. 64861-VL). Protein Identification by nLC-ESI-MS/MS of 1D Gels.  Gelslices of interest were manually excised from 1D-PAGE gels,reduced, alkylated and digested overnight with bovine trypsin.Five microliters of trypsin-digested sample were injected in acapillary chromatographic system hooked on line with an APIQStar PULSAR mass spectrometer following the same condi-tions as described above. Analysis parameters and Mascotsearches were set as described above. Scaffold (version Scaffold-01_06_07, Proteome Software, Inc., Portland, OR) was used tovalidate MS/MS based peptide and protein identifications.Peptide identifications were accepted if they could be estab-lished at greater than 95.0% probability as specified by thePeptide Prophet algorithm. Protein identifications were ac-cepted if they could be established at greater than 99.0%probability and contained at least 2 identified peptides. Proteinprobabilities were assigned by the Protein Prophet algorithm.Proteins that contained similar peptides and could not bedifferentiated based on MS/MS analysis alone were groupedto satisfy the principles of parsimony.  Western Blot Analyses.  Gels were electroblotted on nitrocel-lulose (NC) membranes in Tris-glycine buffer (50 mM Tris, 380mM glycine) and 20% (v/v) methanol, at 4  ° C (60 min, 400 V).NC membranes were blocked by incubation in 5% BovineSerum Albumin (BSA) (w/v) overnight, then washed in 10 mMPBS with 0.15% Tween-20 (v/v) (PBS-T) and probed withprimary Ab’s (mouse monoclonal Ab against Rh, diluted 1:500in PBS; and rabbit polyclonal Ab anti- R  /    subunits of F 1 F o - ATP synthase diluted 1:5,000 in PBS). Incubation was con-ducted for 1 h at 25  ° C under stirring. After extensive washing  with 0.15% PBS-T, binding of Ab was revealed by enhancedchemiluminescence detection system (ECL) (Roche DiagnosticsCorp., Indianapolis, IN) using horseradish peroxidase-conju-gated anti-rabbit or anti-mouse IgG (Amersham Biosciences,Piscataway, NJ), diluted 1:5000 in PBS. Blots were then auto-radiographed onto Hyperfilm ECL (Amersham Biosciences,Piscataway, NJ) and films acquired. Semiquantitive Western Blot Analysis and Quantificationof Identified Proteins.  Quantitative densitometry was per-formed using the ImageJ 1.31v software ( Each ECL band on acquired autoradiography film andthe corresponding whole protein pattern was converted into adensitometric trace, allowing calculations of intensity. Forquantification, densitometric values were compared and resultsexpressed as Relative Optical Density (R.O.D.).  ATP Synthesis Assay.  ATP synthesis was measured by thehighly sensistive luciferin/luciferase method. Assays wereconducted at 37  ° C over 1 min, by measuring formed ATP fromadded ADP. Freshly isolated disks (0.6 mg of protein) wereadded to incubation medium (1 mL final volume) containing:10 mM Tris-HCL (pH 7.4), 50 mM KCl, 1 mM glucose, 10 mMsuccinic acid, 1 mM EGTA, 5 mM MgCl 2 , and the adenylatekinase inhibitor di(adenosine-5 ′ ) penta-phosphate, 0.2 mM(final ampicillin concentration was 40  µ g/mL). Suspension wasequilibrated in order to energize membranes (for 10 min at 37 ° C), then ATP synthesis was induced by addition of 0.3 mM ADP, and 5 mM KH 2 PO 4. . When necessary, incubation mediumcontained either 0.5 mM  N  , N  ′ - dicyclohexylcarbodiimide (DCCD)and 10  µ M olygomycin. When indicated, 10  µ M nigericin 27  wasadded after preincubation. The rate of ATP synthesis wasmeasured by stopping the reaction with 25% (v/v) perchloricacid (PCA). The ATP content of neutralized (with 2 M K  2 CO 3 )and clarified samples was measured using the luciferin/luciferase ATP bioluminescence assay kit CLSII (Roche, Basel,Switzerland), on a Luminometer (Triathler, Bioscan, Washing-ton, DC). ATP standard solutions (Roche, Basel, Switzerland)in the concentration range of 10 - 10 - 10 - 7 M were used forcalibration. Cellular Subfraction Preparations.  All operations werecarried out at 4  ° C. 1. Retinal Mitochondria-Enriched Fractions.  Residual reti-nas after ROS preparation were collected (4 g) and homog-enized in 4 mL of 0.25 M sucrose and 5 mM  N  -2-hydroxyeth- ylpiperazine- N  ′ -2-ethanesulfonic acid (HEPES), pH 7.2, proteaseinhibitor cocktail and 60 mg/mL ampycillin, and centrifugedat 700 g   for 10 min in a Heraeus centrifuge. The pellet wasdiscarded, and the supernatant was centrifuged at 10 900 g   for10 min. The pellet was retained as a mitochondria-enrichedpreparation. Standard Procedures.  Protein concentrations were deter-mined by the bicinchoninic acid (BCA) protein assay fromPierce Biotechnology, Inc. (Rockford, IL). Results and Discussion  Assessment of Purity of the Disk Preparations.  Osmotically intact disks were prepared by Ficoll flotation. 11 To minimizecontamination by RIS structures, ROSs were not homogenized,but burst in distilled water. Disks were collected at the interfacebetween a 5% Ficoll solution and water, after ultracentrifuga-tion. 11 The cellular material that bursts upon 3 h incubationin hypotonic solution is sedimented by the ultracentrifugation.Disks do not burst by simple hypotonic shock: sonication ortreatment with 1% detergents is necessary. Likely, the peculiarosmotic resistance of disk membranes to distilled water is dueto their particular phospholipid composition, whose fatty acidsare highly unsaturated, so that the overall phase is a fluid film. 28 Contamination by vesicles of other nature or by resealed pieces Proteomics of Retinal Rod Disks   research articles Journal of Proteome Research  •  Vol. 7, No. 7, 2008  2657
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