1. Tissue Processing and Protein Extraction
1.1 Extraction With Acetic Acid/Mercaptoethanol
A number of methods have been developed to extract proteins from tissues for proteomics. The AcOH/mercaptoethanol method is well suited for larger tissue specimens, and sufficient amounts of protein can be obtained for multiple experiments. The lyophilized material produced by this method can be stored for long periods of time and can be weighed out in appropriate amounts on a microbalance. The lyophilized material can be re-dissolved in a rehydration buffer of the selected composition, which provides flexibility to perform different types of experiments with the same tissue extract. Using this method, there is no need to separate proteins from other cellular components.
1) Quickly weigh the frozen tissue specimen (typically 0.4–0.5 g of pituitary tissue) and cut it in several pieces; place the tissue pieces in a 50-mL plastic tube.
2) Rinse the tissue three times with 10 mL of ice-cold saline solution (the tissue will have thawed by this time).
3) Add to the tissue 10 mL of the homogenization buffer (chill buffer on ice before use); homogenize with the Polytron homogenizer power set to 6 (keep the mixture on ice during homogenization, alternate 10 s homogenization and 30 s of rest; repeat until complete disruption of the tissue is achieved).
4) After homogenization, sonicate the homogenate for 10 s (keep the homogenate on ice during sonication); repeat this step.
5) Aliquot the homogenate into 1-mL fractions; lyophilize these fractions.
6) Determine protein concentration in the lyophilizate.
7) Store the lyophilizate at –80°C until further use.
1.2 Extraction With TRIZOL Reagent
The TRIZOL-based method is well suited for large tissue specimens. With this method, cellular proteins are separated from RNA and DNA. Prolonged storage of the protein extract may result in problems with subsequent solubilization.
1) Quickly weigh the frozen tissue specimen and cut it in several pieces; place the pieces in a 14-mL plastic tube.
2) Rinse the tissue three times with 5 mL of ice-cold saline solution.
3) Add 4 mL of the TRIZOL reagent per gram of tissue.
4) Homogenize the mixture with the homogenizer power set to 6; (keep the mixture on ice during homogenization, alternate 10 s homogenization and 30 s of rest; repeat this step until the tissue is fully disrupted).
5) After homogenization; sonicate the sample (keep suspension on ice, alternate 10 s of sonication and 30 s of rest; repeat this step three times).
6) Vortex the sample for 4 h at 4°C.
7) Add 0.2 mL of chloroform per 1 mL of TRIZOL reagent used.
8) Centrifuge at 12,000g for 10 min. The mixture will separate into three phases: upper (aqueous) phase, interphase, and lower (organic) phase.
9) Remove the upper (aqueous) phase.
10) To the lower phases (interphase and organic), add 0.3 mL of ethanol per 1 mL of TRIZOL reagent used; mix by inversion.
11) Centrifuge at 2500g for 5 min.
12) Draw off the supernatant, and add to this supernatant a volume of acetone five times the volume of the supernatant solution; mix by inversion.
13) Centrifuge at 12,000g for 10 min. After centrifugation, remove and discard the supernatant.
14) Wash the protein pellet with 95% ethanol containing 0.3 M guanidine-HCl (2 mL per 1 mL of TRIZOL reagent used); repeat three times.
15) Vortex the protein pellet in 2 mL of ethanol, store the protein pellet in ethanol for 20 min, centrifuge at 7500g for 5 min.
16) Remove ethanol and dissolve the pellet in 2–3 mL of IEF rehydration buffer containing no dye.
17) Determine protein concentration.
18) Aliquot the sample and store at –80°C until further use.
2 In-Gel IEF
2.1 Sample Preparation for IEF
1. Weigh out a required portion of the pituitary lyophilizate; or measure out the TRIZOL extract.
2. Add 360 μL of IEF rehydration buffer.
3. Vortex the sample mixture for 1 h; at 20, 40, and 60 min, sonicate the sample for 20 s with the probe sonicator.
4. Centrifuge the sample at 21,000g for 30 min.
5. Take out the IPG strip(s); with a permanent marker, outline on the plastic backing the sections to be cut later.
6. Apply 350 μL of the sample solution into a slot in the DryStrip reswelling tray.
7. Remove the protective cover from the IPG strip and place the IPG strip gel-side down on top of the sample solution.
8. Overlay the strip with 3 mL of oil to prevent evaporation.
9. Rehydrate the IPG strip overnight.
2.2. Isoelectric Focusing
1) Set the MultiTemp circulator to 20°C.
2) Pour 4 mL of oil (cover fluid) onto the cooling plate of the Multiphor II unit; position the DryStrip running tray onto the cooling plate; connect electrode wires to appropriate positions.
3) Pour 10 mL of oil into the DryStrip tray; place IPG strip aligner on top of oil.
4) Cut two electrode paper strips to a length of 110 mm and moisten them with 0.5 mL of water (they should be just damp).
5) Lift the rehydrated IPG strip out of the reswelling tray; rinse the gel surface with water; gently sweep the strip backing over a sheet of paper to remove excess oil.
6) Place the IPG strip into a groove in the strip aligner, gel side up and in the correct orientation.
7) Place the moist electrode paper strips across the cathodic and anodic ends of the IPG strip; the electrode paper strips must be in contact with the gel ends of the IPG strip. Position the electrodes over the electrode paper strips and press them down to come in contact with the electrode paper strips.
8) Pour 70–80 mL of oil into the tray to completely cover the IPG strip(s).
9) Perform IEF according to the following protocol: 0–100 V (gradient over 1 min); 100 V (fixed for 120 min); 100–500 V (gradient over 1 min); 500–3500 V (gradient over 90 min); 3500 V (fixed for 8 h).
10) After completion of IEF, remove the IPG strip, wipe off excess oil, and loosely cover the IPG strip with plastic wrap; store the wrapped IPG strip at –80°C until further processing.
3 Processing of the IPG Strip and Protein Digestion
1. Rinse the IPG strip with 200 mM ammonium bicarbonate for 10 s.
2. Incubate the strip in 10 mL of 200 mM ammonium bicarbonate for 10 min.
3. Use a clean scalpel to separate the strip into gel sections; place each gel section into a siliconized 0.5-mL Eppendorf tube.
4. Dehydrate each section with 50 μL of acetonitrile; repeat twice (see Note 24).
5. Dry the gel sections in a vacuum centrifuge for 30 min.
6. Rehydrate each gel section with 100 μL of ammonium bicarbonate (50 mM) containing trypsin (20 μg/mL).
7. Incubate the samples in a water bath at 37°C overnight.
8. After digestion, centrifuge the samples at 10,000g for 1 min and transfer each supernatant into a clean 0.5-mL tube.
9. To extract the remaining peptides from each gel section, add 70 μL of acetonitrile/water/ trifluroacetic acid (50:45:5; v:v:v) and sonicate the sample in a sonicator bath for 20 min; collect the supernatant and repeat the extraction one more time; combine all extracts from each gel section.
10. Dry the samples in a vacuum centrifuge.
11. Reconstitute the samples in 15 μL of water/0.1% trifluoroacetic acid.
12. Centrifuge the samples at 10,000g for 1 min.
13. Purify the digests with ZipTip tips, following manufacturer's instructions; elute the peptides from the ZipTip with 3 μL of acetonitrile/water (50:50; v:v).
14. Add to each eluate 3 μL of water/1% acetic acid/0.02 % heptafluorobutyric acid.
15. Store the peptide samples at –20°C until analysis.
4 LC-MS/MS
To obtain peptide sequence data, digests from each fraction of IPG strips were analyzed by LC-MS/MS. In this experiment, the peptide mixture was separated by reversed-phase high performance liquid chromatography (HPLC) using a gradient of water/acetonitrile. The peptides eluted from the liquid chromatography column were introduced online into the mass spectrometer and analyzed. The mass spectrometry analysis is performed in a data-dependent mode, with the instrument switching between MS and MS/MS. Typically, full scan MS spectra are acquired to determine the molecular mass of the peptide, and then MS/MS (product-ion) spectra are acquired for several peptide sequence diagnostics. This cycle is repeated throughout the run, resulting in a large amount of MS/MS data for the peptides in a given digest. In this experiment, the mass spectrometer cycles between acquiring a full scan of the mass spectrum and performing five MS/MS scans for the most abundant ions in the mass spectrum scan.
5 Database Searches
MS/MS data are used to search the protein sequence database (Swiss-Prot or NCBInr) to identify proteins in each fraction of the IPG strip. The database search results are manually checked to identify retrieved proteins with significant scores and to remove false positive hits.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.
