A proteomics study of chemically induced cirrhosis in rat liver revealed the mechanism of thioacelamide hepatotoxicity 2

Chapter Materials and Methods Chapter 2.1 Materials Analytical grade reagents were used for the preparations of general reagents Special reagents were obtained from: Sigma Chemical Co Ltd (U.S.A.) α-cyano-4-hydroxycinnamic acid Ammonium carbonate Coomassie Blue R-250 Ethylenediaminetetraacetic acid (EDTA) Phenylmethyl sulfonyl fluoride (PMSF) Thioacetamide (TAA) Trifluoroacetic acid (TFA) All common laboratory chemicals Bio-Rad Laboratories (U.S.A.) 40% acrylamide/ bis solution in 37.5: ratio Ammonium persulfate (APS) Dithiothreitol (DTT) EDTA disodium dihydrate Polyacrylamide gel reagents Broad range protein molecular weight standards TEMED Urea 56 Chapter PD-Quest software Amersham Pharmacia Biotech (Sweden) Agarose (USB) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) Immobilized pH gradient (IPG) strips pH 3-10 IPG buffer J.T.Baker (U.S.A.) Tris(hydroxymethyl) aminomethane (Tris) BDH Laboratory Supplies Glycerol Merck (Germany) Acetic acid Formaldehyde Methanol Silver nitrate Sodium carbonate Sodium thiosulphate Sodium dodecyl sulphate (SDS) 57 Chapter Fluka Chemie (Switzerland) Iodoacetamide (IAA) Thiourea EM Science (U.S.A.) Acetonitrile Boehringer Mannheim (Germany) DNase RNase Promega (U.S.A.) Modified porcine trypsin PIERCE (Rockford, Bedford, MA, USA) Coomassie® Plus Protein Assay 58 Chapter 2.2 Chemical induction of liver fibrosis 2.2.1 Experimental animals 8-9 weeks old male Wistar-Furth rats were purchased from Sembawang Animal Centre, Singapore They were housed separately in an animal room on a 12 h light-dark cycle with free access to food and water These rats were kept for a week for acclimatization prior to thioacetamide (TAA) administration 2.2.2 Preparation of 10% thioacetamide To prepare a 10% TAA solution, 10 g of TAA was weighed and dissolved in 100 ml of autoclaved de-ionized water The container was wrapped up with aluminum foil and stored at 4ºC TAA crystals reformed during storage so just before injection, TAA was redissolved by stirring with a magnetic stirrer 2.2.3 Administration of TAA TAA was administered to the rats via intraperitoneal injection Each rat was weighed before being injected with 300 mg/kg of TAA using the formula below: if the rat weighs N kg, then 10% TAA to inject is x N ml Each experimental rat was injected for times per week, using a 27G 1/2 needle with at least a day’s rest in between injections The rats were divided into groups; each group was treated for 3, and 10 weeks respectively The paired controlled animal received no treatment 59 Chapter 2.2.4 Sacrifice of animals After the final injection, the rats were left untreated for a week to clear the circulating TAA from their systems before sacrifice During sacrifice, the rats were anesthetized with ether in a hood before surgery 2.2.5 Surgical removal of liver tissue During sacrifice, a consistent lobe of liver was surgically removed, sliced into small pieces, stored in ml cryo-vials and immediately snapped-frozen in liquid nitrogen Then, the tissues were transferred to -152ºC freezer for storage 2.3 Analysis of liver histopathology Following fixation, the liver was embedded in paraffin and sectioned 4-µm sections were mounted on slides and stained with Masson Trichrome stain The tissue sections were examined blind by a trained liver pathologist Each section was graded under light microscopy according to the amount of positively stained collagen The degree of liver fibrosis was graded according to a modified scoring system below as reported by Ruwart et al (Ruwart et al, 1989) Ten different fields of the liver were examined and scored In those liver sections with obvious nodularity, each field was examined at x4 magnifications In those liver 60 Chapter sections with little or no nodularity, each field was examined at x20 magnifications Each animal would have a total minimum score of or maximum score of Table 2-1 The scoring scheme used in the assessment of liver histopathology Score Description No collagen or fibrosis Slightly increased collagen Definite increase, without septa, generally seen as small stellate expansions of collagen from the central zones or pericentrally in the lobules Septa are identified as linear collagenous extensions from microscopic landmarks, usually terminal hepatic venules Definite increase with incomplete septa (those septa which not interconnect with each other so as to divide the parenchyma into separate fragments) Definite increase with complete septa but thin septa (those septa which interconnect with each other so as to divide the parenchyma into separate fragments) 61 Chapter 2.4 Preparation of liver tissue lysate for 2DE Liver tissues were kept in liquid nitrogen upon retrieval from the freezer They were ground with a pestle and mortar in liquid nitrogen until finely powdered For safety purpose, the pounding was performed in a class II cell culture hood with full laminar flow ventilation Subsequently, lysis buffer consisting of M urea, M thiourea, 4% 3-[(3cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), 40 mM tris(hydroxymethyl aminomethane (Tris), mM PMSF (Sigma), 0.5 mg/ml DNase, and 0.5 mg/ml RNase was then added to the powder and the grinding continued until the buffer mixed well with the tissue in powder form Finally, the mixture was left to thaw Tissue lysate was then carefully taken up with a pipette into an Eppendorf tube and subjected to ultracentrifuge for h at 100, 000 x g The resulting supernatant was retrieved and kept in -80ºC deep freezer 62 Chapter 2.4.1 Preparation of lysis buffer Lysis buffer is prepared as the following: Table 2-2 Composition of lysis buffer for 2-D gels Buffer components Urea Thiourea CHAPS Tris-base H2O Amount/Volume added Concentration 21.021 g 7M 7.612 g 2M 2.0 g 4% w/v 0.2422 g 40 mM 50 ml - Aliquots of ml each were transferred to an Eppendorf tube and stored at -20°C 2.5 Protein estimation Protein concentration was estimated using Coomassie® Plus assay kit This kit is compatible with the high concentration of urea and thiourea used in the lysis buffer Concentration of protein was estimated by measuring the adsorbance at 595 nm, using BSA to establish a standard curve 63 Chapter 2.6 Two-dimensional gel electrophoresis (2-DE) 2.6.1 First dimension - isoelectric focusing (IEF) 2.6.1.1 Rehydration of dehydrated IPG strips For rehydration, precast 18 cm long, pH 3-10 non-linear, dry immobilized pH gradient strips (IPGs) were immersed overnight in rehydration buffer containing M urea, M thiourea, 4% CHAPS, 20 mM DTT and 5% IPG buffer (pH 3-10) This process was first performed by pipetting 340 µL of the rehydration buffer into the ceramic strip holders Then, the protective plastic layer of the IPG gel was removed and the IPG gel was placed onto the strip holder with the gel-coated surface facing down, so that the gel surface was in contact with the rehydration buffer To prevent uneven rehydration, trapped air bubbles were smoothened carefully with a clean forceps This is followed by covering the gel with a layer of paraffin oil and a plastic cover so as to prevent exposure of the rehydration buffer to air This is important because crystallation of the highly concentrated urea might occur due to evaporation of the buffer Rehydration should take at least 10 h and it is usually performed overnight Rehydration is an important step because it ensures that the dehydrated strips were rehydrated so that they are able to adsorb solubilized protein samples during isoelectric focusing (IEF) 64 Chapter 2.6.2.2 Preparation of SDS equilibration buffers The equilibration buffer was prepared as follows: Table 2-5 Composition of buffer used for equilibration of IPG strips pH 6.8 buffer Amount/volume pH 8.8 buffer Amount/volume Final Concentration Tris-HCl (pH 6.8, 1.0 M) 50 ml - 50 Mm Tris-HCl (pH 8.8, 1.5 M) - 33.33 ml 50 mM 360.63 g 360.63 g 6M 300 ml 300 ml 30% 20 g 20 g 2% trace trace - Top up to L Top up to L Stock Solutions Urea Glycerol SDS Bromophenol blue H2O 68 Chapter 2.6.2.3 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SDS-PAGE is a rapid method for protein separation, quantification, and characterization SDS, being an ionic detergent, binds to proteins at a uniform negative charge density and denatures them Thus, SDS-bound polypeptide is separated in the gel according to its molecular weight alone SDS-PAGE with the tris-glycine system (Laemlli, 1970) was chosen as the second dimension for its unsurpassed resolution and ease of use 10% T SDS-PAGE gels were cast as the following composition: Table 2-6 Composition of SDS-PAGE used for second dimensional separation Stock solution Volume (ml) 40% acrylamide/ bis solution in 37.5: ratio 50.0 1.5 M Tris-HCl, pH 8.8 50.0 Deionized water 97.0 10% SDS 2.0 10% APS 1.0 TEMED 0.125 69 Chapter 2.6.2.4 Preparation of sample loading buffer Protein samples need to be solubilized and denatured before SDS-PAGE This was accomplished by dissolving protein samples in the sample loading buffer 2x sample loading buffer was used for liquid sample and was prepared as the table below Aliquots of ml each were transferred to Eppendorf tubes and stored in freezer at -20°C Table 2-7 Composition of sample loading buffer for SDS-PAGE Ingredients Volume (mL) MilliQ Water 1.30 1.0 M Tris-Cl pH 6.8 1.20 Glycerol 2.00 10% (w/v) SDS 4.00 Bromophenol blue (0.1%) 1.00 β-mercaptoethanol 0.50 Total 9.50 70 Chapter 2.6.2.5 Preparation of electrode buffer Tris-glycine buffer was the electrode buffer used in our SDS-PAGE experiments It was prepared as follows: Table 2-8 Composition of electrode buffer used for SDS-PAGE Ingredients Amount/Volume 15.00 g Tris Glycine 1.0 g of SDS MilliQ water 2.6.2.6 72.00 g 5.00 g Top up to L Molecular weight standards Broad range standard molecular weight markers (Bio-Rad) used included rabbit skeletal muscle myosin (200 000 Da), E Coli beta galactosidase (116, 250 Da), rabbit muscle phosphorylase b (97 000 Da), bovine serum albumin (66 200 Da), hen egg white ovalbumin (45 000 Da), bovine carbonic anhydrase (31 000 Da), soybean trypsin inhibitor (21 500 Da) and hen egg white lysozyme (14 400 Da) 71 Chapter 2.6.2.7 Treatment of protein molecular weight markers Molecular weight markers were mixed with 2x SDS sample loading buffer at a ratio of 1:1 and then heated at 95°C for prior to application About µl of this mixture was dispensed carefully onto a piece paper application strip before being transferred to the second dimension 2.6.2.8 Transfer of IPG strip to the second dimension After SDS equilibration, the IPG strip was rinsed in purified water and blotted dry before being transferred to the second dimension by gently laying the strip on top of a SDS-PAGE gel Protein molecular weight markers loaded on a piece of application strip was inserted at one end of the strip This was followed by gently dispensing 0.75% agarose to seal the IPG strip and the paper strip to the second dimension 2.6.2.9 SDS-PAGE running conditions The second dimensional gel was performed using a Bio-rad Protean IIxi gel set with the power pack set at a constant current of 30 mA per gel and the system was cooled to 10°C with a Pharmacia LKB – MultiTemp II cool water circulator 72 Chapter 2.7 Image visualization and analysis To detect and visualize any protein spots, the 2-D gels were stained with silver After gel electrophoresis, the transparent gels were recovered and immediately kept in fixing solution prior to staining to visualize protein spots Silver staining was performed according to Shenchenko et al (Shenchenko et al., 1996) Silver stain can detect up to nanogram of proteins and hence is suitable for staining 2-D gels 2.7.1 Silver staining procedure 2-D gels were first immersed in fixing solution for 30 before washing with 50% methanol in water for another 30 Then, the gels were washed again with water for 60 followed by sensitization with 0.02% sodium thiosulphate for After the gels were rinsed with water twice for each, they were incubated with chilled 0.1% w/v silver nitrate for 40 at 4°C After discarding the silver nitrate and rinsing with changes of water for each, the gels were developed in 0.04% formalin (35% formaldehyde in water) in 2% sodium bicarbonate When the desired image intensity was attained, the developing solution was discarded and the gel incubated with 1.46% EDTA disodium dihydrate for 10 to stop the development The silver stain procedure was completed with rinses of water for each 73 Chapter 2.7.2 Preparation of silver stain reagents Reagents for silver stain were prepared as follows: Table 2-9 Composition of reagents used in silver stain Solutions Ingredients Composition methanol MilliiQ water 2.50 L 5.00 L 1.0 g MilliQ water Top up to 5.0 L Silver Nitrate 2.5 g (0.1%) MilliiQ water Top up to 2.5 L sodium carbonate 100 g (2%) formalin 0.04% MilliiQ water Top up to 5.0 L EDTA-Na2.H2O Stop Solution 2.50 L sodium thiosulphate Developing Solution 5.00 L TOTAL Silver Nitrate 2.25 L Methanol Sodium Thiosulphate 250 mL (5%) TOTAL 50% Methanol acetic acid MilliQ water Fixing Solution 2.50 L (50%) 73 g MilliiQ water Top up to 5.0 L 74 Chapter 2.7.3 Image acquisition and analysis Stained gels were scanned using a Molecular Dynamics Personal Densitometer SI (Sunnyvale, CA, USA) at 1200 dots per inch (dpi) Images were saved and archived as gel format for subsequent image analysis Analysis of 2-D gel images for the detection of differentially expressed proteins was performed in steps First, a visual inspection of the representative gels (controls compared to the treated samples) was performed to identify protein spots that were markedly different in their expression levels This process is also called the “eye-balling” approach Then, a more detailed analysis of all the triplicates gels were performed using the image analysis software, PDQuest (Bio-Rad) 75 Chapter 2.7.3.1 Eye-balling approach In this analysis, visual inspection of the representative gels was performed The representative gel is the “best gel” chosen from the triplicate gels to represent a liver sample It was chosen based on good image resolution, spot sharpness and highest number of spots For comparison, the best gel of a control sample was paired up with its respective treated counterpart to identify the proteins spot with markedly different intensities To avoid ambiguity, only spots that were present/absent or prominently different in intensities were chosen Subsequently, all the control representative gels were collectively analyzed for spots that were consistent among the same controls This process was repeated for all the best gels of the treated samples Labeled spots that were present at least in out of pairs of the representative gels are chosen and labeled on a “Master Gel” 76 Chapter 2.7.3.2 PDQuest® (software analysis) As the second step, PDQuest software was used for the subsequent image analysis With the reference of the master gels from visual inspection, we confirmed, using PDQuest, the reproducibility of the spots in all the triplicate gels In PDQuest®, a representative or reference gel was first selected Then, spot detection parameters were optimized on this reference and applied to other member gels After one or more sets of detection parameters were created and saved, it was applied to detect spots in all gels After detecting spots on all gels, spots were compared across gels A match set was created Match-sets are sets of gels that have been grouped together for the purpose of qualitative and quantitative comparison Spots on gel were then compared to spots on every other gels in the match-set At the same time, the intensity-volume of the spots was determined in normalized average values A student’s t test at 5% significance level was later performed as an analysis set 77 Chapter 2.8 Identification of protein spots Identification of protein spots of interest was performed using matrix assisted laser desorption/ionization – time of flight mass spectrometry (MALDI-TOF MS) First, the proteins spots were excised and digested in-gel with trypsin The peptides were then extracted from the gel pieces and subject to mass-spectrometric analysis 2.8.1 Excision of protein spots Protein spots of interest were excised manually with a homemade plastic plunger and transferred to a 96-well polypropylene microtitre plate To avoid contamination from keratin, a new pair of gloves was worn each time when performing trypsin digest Each excised spot was washed with 150 µl of 2.5 mM ammonium bicarbonate (NH4HCO3), in 50% acetonitrile overnight The plate was sealed with an adhesive film and stored at 4°C Alternatively, the excised spots were washed with 150 µl of 2.5 mM ammonium bicarbonate (NH4HCO3), in 50% acetonitrile for times of 20 each This washing step was critical as it allowed more efficient enzyme digestion and removal of SDS 78 Chapter 2.8.2 Reduction and alkylation of excised protein spots The solution was replaced with a fresh aliquot and plates were incubated with shaking for 20 at 37°C The washing solution was then aspirated and gel spots were dried in a SpeedVac (Savant Instruments Inc., Halbrook, NY) for 20 The gel spots were reswollen in 20 µl of solution containing 10 mM DTT/100 mM NH4HCO3 and incubated at 56°C for h The solution was subsequently replaced with 20 µl of solution containing 55 mM IAA/100 mM NH4HCO3 and incubated in the dark, at room temperature for 45 min, with occasional vortexing Then, the liquid phase was aspirated and the gel was then washed in 100 µl of 100 mM NH4HCO3 solution for 10 min, followed by a 2-min sonication Subsequently, the solution was removed and the gels were dehydrated in 100 µl of 100% acetonitrile for 10 min, again, followed by a 2-min sonication The above wash and dehydration steps were repeated once Finally, the batch of solution was removed and gel spots were dried in a SpeedVac 79 Chapter 2.8.3 In-gel trypsin digestion After reduction and alkylation, the excised gel spots were each digested with 10 µl of 0.02 µg/µl sequencing grade modified porcine trypsin in 25 mM NH4HCO3, pH 8.5 and incubated at 37°C overnight with shaking 2.8.4 Extraction of peptides from gels To extract trypsin-digested peptides from the gel plug, 10 µl of 0.1% trifluoroacetic acid (TFA) in 50% acetonitrile was added to each well followed by sonication for 20 in an ultrasonic water bath (Crest Ultrasonics, Tenton, NJ, USA) The resulting solution contained peptides from proteins of interest and was used directly for mass determination with a MALDI-TOF MS 2.8.5 Preparation of matrix solution A matrix is a small organic acid used to co-crystallize with the extracted peptides The laser energy from the laser source of the MALDI-TOF MS was transferred to the matrix-coated peptide Then, the peptides were forced to travel in the TOF tube for mass determination The matrix was prepared by dissolving 10 mg of α-cyano- hydroxycinnamic acid in ml 0.1% TFA/50% acetonitrile 80 Chapter 2.8.6 Application of matrix and peptide samples µL of extracted peptide sample was spotted onto a stainless steel MALDI sample plate followed by µL of matrix solution Samples were left to air-dry for subsequent MS analysis 2.8.7 Mass spectrometry Mass spectrometric analyses were performed using a PerSeptive Biosystems Voyager-DE STR MALDI-TOF-MS with the following specifications: The MS automatic data acquisition was performed in delayed extraction, reflectron mode, with accelerating voltage of 20, 000 volts; grid voltage, 74.5%; guide wire, 0.01% and extraction time of 100 nsec Laser intensity was set at 2000, with 50 shots per spectrum The mass range was set between 700-3000 Da The porcine trypsin auto-digest peptides (842.5090 Da and 2211.1040 Da [M+H+] ions) were set as internal calibration standards 2.8.8 Baseline correction, noise filtering, calibration and deisotoping Mass spectra were then corrected for baseline, noise filtered and monoisotopic peaks were chosen by de-isotoping After that, the porcine trypsin auto-digest peptides (842.5090 Da and 2211.1040 Da [M+H+] ions) were used to calibrate the spectra 81 Chapter 2.8.9 Database searching and identification of proteins Database searching was performed with the AutoMS-Fit software (Protein Prospector; UCSF, San Francisco, CA, USA) by submitting the results to SWISS-PROT and NCBI non-redundant protein databases to match the peptide mass fingerprints (PMF) (Henzel et al., 1993; James et al., 1993; Mann et al., 1993; Yates et al., 1993) Processing tools selected from Proteomics Solution 1TM were baseline correction, noise removal and peak deisotoping 25 of the most intense peaks were selected for MS-fit analysis after filtering of common peaks All searches were performed using a mass window between 1000 Da and 100 000 Da which encompasses both human and mouse sequences The search parameters allowed for oxidation of methionine, N-terminal acetylation, carboxyamidomethylation of cysteine, and phosphorylation of serine, threonine and tyrosine Manual MS-Fit was performed after auto-MS-Fit to double check the data 82 ... stirring with a magnetic stirrer 2. 2.3 Administration of TAA TAA was administered to the rats via intraperitoneal injection Each rat was weighed before being injected with 300 mg/kg of TAA using the. .. food and water These rats were kept for a week for acclimatization prior to thioacetamide (TAA) administration 2. 2 .2 Preparation of 10% thioacetamide To prepare a 10% TAA solution, 10 g of TAA was... placing a sample loading cup carefully near the acidic end of the IPG gel 20 µl of rehydration buffer was carefully pipetted into the loading cup to test for any leakages, and was discarded later