Pollution of the marine environment is a growing concern because it can affect humans and other natural species. The application of proteomic methods to monitor marine pollution is a novel approach to assess the ecological impacts of environmental pollution. Aquatic organisms living along the shoreline and in bays are particularly exposed to a variety of pollutants, including peroxisome proliferating pollutants (PPPs). However, both specific peroxisome reactions and common biomarker reactions can be affected by biotic or abiotic factors. This drawback can be overcome by utilizing proteomics technology to evaluate hundreds of proteins simultaneously in two experiments. Applying this technique, we analyzed the mussel function by bidirectional electroswimming .) proteome of the digestive gland peroxisome and compared bidirectional electrophoretic profiles of contaminated samples and controls. Up- and down-regulated proteins constitute a protein expression signature (PES) tag associated with exposure to peroxisome proliferation contaminants. This method yields highly reproducible profiles and can be applied to laboratory or field experiments.
Written by Martin, this experiment is from "Environmental Genomics Lab Guide".
Operation method
A proteomic approach to the risk assessment of peroxisome proliferating contaminants in the marine environment Move I. Materials 1. Animals Mussels (Lmk, 35-45 mm long) or purple mussels (edwfe) with a shell length of 5 cm were collected at different meter sample sites at different times of the year at low tide. One of the sampling sites was a documented clean area where animals were collected as a control. During the isolation process all buffers need to be freshly prepared and operated in an ice bath. (1) 20 % (V/V) TCA (trichloroacetic acid)/cold acetone, 0.07 % (3-phosphoethanol). Store in a refrigerator at -20°C. (2) 1 000 % acetone. Store in a 20°C refrigerator. (1) Solubilization buffer: 7 mol/L urea, 2 mol/L sulfur, 2 % CHAPS, 0.5 % TritonX-100, 1% β-mercaptoethanol, l % Pharmalyte, 1 % DTT. 5 % TritonX-100, 1% β-mercaptoethanol, l % Pharmalyte, 1 % DTT. (2) Rehydration buffer: 8 mol/L urea , 2 % CHAPS, 15 mmol/L D T T , 1 % p-mercaptoethanol, 0 . 2 % Pharmalyte0 . 2 % Pharmalyte0 (3) 0-95 mol/L IAA (iodoacetamide). (1) Equilibration Buffer (EQ): 6 mol/L urea, 50 mmol/L T ris, 30 % glycerol, 2 % SDS, Caulmers Brilliant Blue, pH 8. 8. (2) EQ contains 1 % DTT. (3) EQ contains 4% IAA. These devices are best performed with precast gels and standard types of 12.5 % Tris-HC1. If SDS-PAGE gels are prepared in the laboratory, it is recommended to use Icm-thick gels. (1) Dissolve 0.5 % agarose in electrophoresis buffer. (2) Electrophoresis buffer (5 X ): 125 mmol/L Tris, 960 mmol/L Glycine, 0 . 5 % SDS, pH 8.3. (1) Fixation solution: methanol-acetic acid-water (45 : 1 : 54) (2) Staining solution: 1 7 % ammonium sulfate, 3 4 % methanol, 3 % phosphoric acid, 0 . 1 % ( W/V ) Kaumas Brilliant Blue G250. In this method it is possible to obtain PES consisting of proteins whose expression is altered by exposure to a contaminated environment.In field experiments, the selection of an uncontaminated area as a control area is necessary to obtain reliable results. Ideally, animals collected from control and experimental areas should be exposed to the same biotic (age, shell size, etc.) and abiotic conditions (salinity, pH, temperature, etc.). Tissue homogenization and isolation of subcellular fractions by iodixanol density gradient centrifugation were carried out according to established methods (2 1 ) with minor modifications. The major subcellular fractions were named according to the terminology used by Volkl and Fahimi (22). Thus, the total homogenate is designated A, the heavy mitochondrial fraction is called B, the light mitochondrial or peroxisome-rich fraction is D, the cytoplasmic fraction is E, and the microsomal fraction is F (Fig. 1). (1) Isolation of 50 digestive glands of mussels (~5 g). Experiments were performed simultaneously in all sampling areas. (2) Add ice HM buffer at 3 mL/g (wet weight) and grind the tissue into small flakes in a tissue grinder. (3) Grind three times at low speed with a loosely fitting grinding rod to obtain a tissue homogenate, and transfer the homogenate to a test tube. (4) Centrifuge at 70 g in a low-speed freezer centrifuge for IOmin to remove debris, unbroken cells and most nuclei. (5) Pipette the supernatant, add 2 mL/g of ice HM buffer to the precipitate, and repeat the homogenization and centrifugation. (6) Mix the first and second supernatants as component A. (7) Centrifuge fraction A IOmiiu at 1950 g in a high-speed cryo-centrifuge. (8) After centrifugation, the supernatant is slowly poured out and centrifuged again at 1,950 g for 8 min. The final precipitate consists of the main part of mitochondrial fraction B, and the supernatant is fraction C. The mitochondrial fraction B is the main part of the supernatant. (9) The supernatant component (component C) is centrifuged at 39 OOO g for 45 min and the supernatant including cytoplasm (component E) and microsomes (component F) is removed; the precipitate is then dissolved in 2 mL of ice HM buffer using a glass rod. The precipitate included fractions rich in peroxisomes and light mitochondria, i.e., D. The precipitate was then dissolved in 2 m L of ice HM buffer using a glass rod. (10) Carefully place I mL of component D on top of the gradient solution. The gradient solution consisted of 6 mL of 2 8 % iodixanol (V/V ), 5 mmol/LMOPS, 0 . 1 % ethanol, lmmol/L EDTANa4 solution (pH 7.3, density 1.16 g/mL) and I mL 50 % iodixanol (V A O, 5 mmol/L MOPS, 0.1 % ethanol, I mmol/L EDTANa4 solution (density I.27 g/mL). The peroxisome-rich fractions will be obtained between 28 and 50 % iodixanol solution. (11) Biochemical methods can be used to evaluate the peroxisome-rich fractions obtained. It is important to try to ensure that peroxisome fractions extracted from different sampling points have the same quality. Otherwise, contamination with other proteins adulterated in any sample will interfere with the next step in the analysis. The activities of the following marker enzymes can be measured during isolation: catalase (CAT) in peroxisomes, succinate dehydrogenase (SD) in mitochondria, and acidic phosphatase (AP) in lysosomes (23). Proteins (1) To precipitate the sample, mix 2 0% TCA (0.07% |3-mercaptoethanol in cold acetone) by I X volume with I X volume of sample. The TCA solution must be freshly prepared. The final concentration of TCA is 10 %. (2) Precipitate for at least 45 min at 20°C, stirring every 15 min (see Note 3). (3) Centrifuge at 12 000 g for 10 min in a refrigerated centrifuge. (4) Remove the supernatant and centrifuge at 12 OOOg to remove the remaining TCA, which would otherwise affect the pH. (5) Wash the precipitate with I mL of cold acetone (containing 0.0 7 % |3-mercaptoethanol) and mix well. Then centrifuge at 12,000 g for lOmin at low temperature and repeat if the supernatant is still yellow. (6) Remove the supernatant and shake off the acetone by centrifugation at 12,000 g. This reduces the drying time. This reduces the drying time. (7) Open the cap of the tube and dry it at room temperature for 20 to 30 min. You can cover the mouth of the tube with tin foil or the like to avoid contamination by dust or other things. (1) Add pro-dissolution buffer to the precipitated sample. Add a volume of pro-dissolution buffer equal to one-half of the isoelectric focusing sample volume. Depending on the length of the IPG strips, the following volumes are recommended: 7 cm strips, 125 uL; 1 cm strips, 185 uL; 18 cm strips, 250 uL. 1 uL of 0.01% (W/V) CBB is added and shaken for 15 minutes at room temperature. Add 1 uL of 0.01 % (W/V) CBB and shake for 15 min at room temperature. (2) Add O.95 mol/L IA A to a final concentration of 30 mmol/L. Shake at room temperature for 15 min. This solution should be freshly prepared. (3) Add rehydration buffer (i.e., half of the remaining volume) and shake for 30 min at room temperature. (4) Centrifuge at 5000 g for lOmin at room temperature. (5) Determine protein concentration. To determine protein concentration by any method such as the Bradford method (24 ) or the Smith method (25 ), it is recommended that only 1 to 2 uL of sample be used. (6) If the sample cannot be used immediately, it is recommended that it be stored in the refrigerator at 4°C. The following is an example of using Bio-Rad's Portean IEF cell. (1) Pour the supernatant into the focusing disk and place the IPG adhesive strip on top of it, taking care that there are no air bubbles under the strip. (2) Isoelectric focusing method: For 11 cm IPG strips: passive rehydration for 12-15 h, rapid pressure rise. Step 1: 250V for 15 min. Step 2: 8000V 2. 5 h. Step 3: 8000V until 35 000 V-h is reached (45 OOOV-h if necessary). Rehydrate and add mineral oil to cover the tape. (3) Immediately after isoelectric focusing, IPG tape can be equilibrated or stored in a 20°C refrigerator. (4) Equilibrate IPG film in equilibration solution containing 1 % DTT for 15 min at room temperature on a shaker. (5) Equilibrate IPG strips on a shaker for 15 min at room temperature in a new equilibration solution containing 4% IAA, which should be prepared fresh. The following procedures are based on the use of Bio-Rad's Criterion SDS^PAGE cell or Dodeca cell and Criterion gel (see Note 4, Note 5). (1) Place the IPG strip on top of the SDS--PAGE gel, making sure there are no air bubbles between them. (2) Add 0 .5 % agarose (dissolved in electrophoresis buffer) to the gel. 5 % agarose (dissolved in electrophoresis buffer) to the strip and seal the strip. (3) This process needs to be carried out in a cold room with a magnetic stirring bar to ensure that the electrophoresis buffer is maintained at the same temperature. The electrophoresis buffer temperature should be the same or lower than room temperature. (4) For Ilcm gels, the gels are kept at 120V until the CBB reaches the bottom of the gel. For 18 cm gels, a constant current should be used. Use 16 mA for the first 30 min, then adjust the current to 24 mA until the CBB reaches the bottom. (1) Fix in fixative for 30 min. (2) Stain in Coomassie Brilliant Blue for 12 to 18 min. (3) Decolorize in methanol for 2~3 min. (4) Decolorize in water for 12 h or until the background is clear. It is better to change the water several times so that the background is cleaner. The gel is then kept in a cold room until it is scanned and analyzed or the protein is identified by mass spectrometry (Figure 3, Figure 4). (1) Scanning can be done with the Image Scanner from Amersham Biosciences. (2) Data were analyzed by Image Master Platinum 6.0 from Amersham Bioscience or other appropriate software on the market. (3) Image analysis included point detection, point quantification, normalization, background removal, and point matching followed by statistical analysis. (4) The amount of each protein point is expressed by the volume of the point, defined as the sum of the densities of all the pixels that make up the point. (5) To correct for differences in CBB staining and to reflect differences in volume between points, the volume of a point was further expressed as the percentage of the volume of that point over the volume of all points on the gel. All points between different samples were analyzed for significance by this percentage value. The 2-DE profiles of the samples from the contaminated area can be compared with the control. In order to identify those spots with significantly altered expression (up- or down-regulated), an i-test was performed on both samples and only protein spots with a significance of 95 % or higher were considered. Caveat (1) Leupeptin, peptidase, e-aminohexanoic acid, and DTT reservoirs are dissolved in water. PMSF is a hazardous substance and 100 mmol/L reservoir solution is prepared with isopropylacetone.(2) To prepare a solubilization and hydration buffer, it is recommended that urea be dissolved in a small amount of MilliQ water at room temperature, followed by the addition of thiourea. The urea-thiourea solution is washed through a 10 mg/mL ion exchange resin. After shaking, the solution is allowed to stand for IOmin, and the supernatant is removed with a pipette tip.(3) In the precipitation of proteins by TCA, the yield varies depending on the protein concentration. The best results were obtained at a protein concentration of about 1 mg/mL.(4) When performing SDS-PAGE electrophoresis, make sure that there are no air bubbles between the IPG adhesive strip and the adhesive surface. Another method is to dunk the agar on the SDS^PAGE first, and then slowly place the IPG adhesive strip on it.(5) When performing SDS-PAGE electrophoresis, if the electrophoresis buffer is very cold, the process will be much slower; if the electrophoresis buffer is too hot, the gel will electrophoresis discontinuously. For more product details, please visit Aladdin Scientific website.
concentrations were determined by the Bradford method or other relevant methods (24). Alternatively, different commercially available polyclonal antibodies can be applied for protein gel blot analysis using the classical chemiluminescence method (Fig. 2).
