Substantial equivalence (proteomics) experiments
Wheat, as one of the major global crops, is processed into a range of food products for consumer consumption. Therefore, it is of great importance to determine the effects (intended and unintended) of transgenic technology on wheat and whether transgenic lines are substantially equivalent to conventionally bred lines. Proteomic analysis is one way to address these questions. The source of this experiment is "A Guide to Transgenic Technology and Field Identification Experiments in Wheat Crops" [English] H.D. Jones P.R . Hewry Editor-in-chief.
Operation method
Substantial equivalence (proteomics)
Materials and Instruments
ESI-MS MALDI - MS Move It is notoriously difficult to ensure the reproducibility of bidirectional polyacrylamide gel electrophoresis within and between laboratories. In our laboratory, strict adherence to standard operating procedures (sop ) is mandatory to ensure that reproducible separations can be obtained by different operators. An example of a 2D gel profile of a wheat white flour sample is shown in Fig. 16.1. The Tris-CaCl2 extraction method was not used to extract "total protein" but rather a representative sample of proteins in wheat flour other than gluten. If total protein is extracted, the high content of gluten may mask other less abundant proteins. The same problem may occur with other tissues that contain high abundance of proteins, in particular the enzyme ribulose diphosphate (Rubisco), which is present in green tissues. For more product details, please visit Aladdin Scientific website.
Extraction Buffer
SDS- Polyacrylamide Gel Electrophoresis
We offer three approaches. The first is an extraction method characterized by the use of Tris-CaCl2 extraction, which was used in our laboratory for a large-scale proteomic study of the substantial equivalence of transgenic (GM ) and non-transgenic (non-GM ) wheat. The second and third extraction methods are widely used in the literature and can be applied to a variety of different tissues.
3.1 Protein extraction
3.1.1 Extraction of white flour proteins using Tris-CaCl2 buffer (cf. [ 11 ] )
( 1 ) 2 g of white flour was added to 5 ml of pre-cooled (4°C) extraction solution and stirred at 4°C for 30 min.
( 2 ) Centrifuge at 10000 g for 30 min at 4℃ to remove insoluble matter.
( 3 ) Retain the supernatant in liquid nitrogen and freeze rapidly, divide into small portions and store at -80℃ for use.
( 4 ) Determine the protein content by Bradford method, using BSA as standard.
3.1.2 Extraction of white flour or green tissue proteins by acid/acetone precipitation (refer to [ 4 ] )
3.1.3 Flour
( 1 ) In a 50 ml Falcon centrifuge tube, suspend 5 g of flour in 20 ml of solution I, vortex at maximum speed for 2 min, and let stand at -20℃ for 1 h. The solution should be mixed with the flour and the flour should not be used in the extraction.
( 2 ) Centrifuge at 15000 g for 10 min at 4°C.
( 3 ) Discard supernatant and vortex with 5 ml of Solution II to resuspend the precipitate.
( 4 ) Centrifuge at 15000 g for 10 min at 4°C.
( 5 ) Repeat steps 3 and 4 twice.
( 6 ) Discard the supernatant and dry the precipitate in a fume hood under a continuous stream of nitrogen (ensuring no loss of sample).
( 7 ) Resuspend 40 mg of dry sample in 1 ml of pH-appropriate rehydration buffer, vortex for 5 min, and centrifuge at 13,000 g for 10 min at room temperature.
( 8 ) Retain supernatant and dispense in small portions. Freeze rapidly in liquid nitrogen.
( 9 ) Store at -80°C.
( 10 ) Determine the protein concentration of the small sample before use.
3.1.4 Green Tissue
( 1 ) Grind snap-frozen leaves in liquid nitrogen using a pre-cooled mortar and pestle.
( 2 ) Suspend 2 g of leaf powder in 20 ml of solution I, vortex at maximum speed for 2 min, and precipitate at -20°C for 1 h. The solution should not be used as a liquid.
( 3 ) Centrifuge at 15000 g for 10 min at 4°C, discard supernatant.
( 4 ) Add 5 ml of Solution II, vortex and resuspend the precipitate, centrifuge at 15000 g for 10 min at 4℃.
( 5 ) Repeat Step 4 twice and dry the precipitate in a fume hood under a continuous stream of nitrogen to ensure no loss of sample.
( 6 ) Resuspend 20 mg of dry sample in 1 ml of pH-appropriate rehydration buffer.
( 7 ) Vortex for 5 min and centrifuge at 13000 g for 5 min at room temperature.
( 8 ) Retain supernatant and dispense in small portions. Freeze rapidly in liquid nitrogen and store at -80°C for later use.
( 9 ) Determine the protein concentration of small samples before use.
If the extracted protein is contaminated with chlorophyll, try PEG/MgCl2 precipitation (see Note 3).
3.1.5 Osborn hierarchical separation (see [ 12 ] )
( 1 ) Add 100 mg of flour to 1 ml of water-saturated n-butanol and stir for 1 h at room temperature.
( 2 ) Centrifuge at 5000 g for 10 min at room temperature and remove the supernatant. This is an optional step for "degreasing".
( 3 ) Extraction of "albumin/globulin": precipitate (from step 2) with 1 ml of 0.5 mol/L NaCl, stir at room temperature for 1 h. Centrifuge as above, retain supernatant and repeat extraction.
( 4 ) Combine the supernatants, and dialyze with 5% (V/V) acetic acid at 4℃ for 48 h, during which the dialysate should be changed at least 4 times, 5 L each time.
( 5 ) Extraction of "Alcohol soluble proteins": precipitate (from step 3) is added to 1 ml of 70% (V/V) ethanol and stirred at room temperature for 1 h. Centrifuge as before. Retain the supernatant and repeat the extraction.
( 6 ) Combine the supernatants and dialyze well for 48 h at 4°C with 5% (V/V) acetic acid (at least 4 changes of 5 L solution each).
( 7 ) Extraction of "gluten": precipitate (from step 5) with 1 ml of 50% (V/V) propanol/2% (V/V) β-mercaptoethanol/1% (V/V) β-mercaptoethanol. (V/V) Acetic acid and stir at room temperature for 1 h. (Note: DTT can be used directly in place of β-mercaptoethanol if required). Centrifuge as previously described, retain the supernatant and repeat the extraction. Combine the supernatants, 5% (V/V) (V/V) acetic acid (at least 4 changes of 5 L of solution each) and dialyze for 48 h at 4°C.
( 8 ) After dialysis, lyophilize all solutions.
( 9 ) The final precipitate (from step 7) can be extracted with 1 ml of "Total Protein Extraction Buffer" (IEF rehydrated): mix and extract for 1 h at room temperature, centrifuge as before and keep the supernatant. Divide into small portions, freeze rapidly in liquid nitrogen and store at -80°C.
3.2 Isoelectric Focusing (IEF)
Immobilized pH gradient (IPG) adhesive strips are available in a variety of lengths (e.g., 7 cm, 13 cm, 18 cm, 24 cm) and in different pH ranges. IPG adhesive strips are often hydrated overnight (16 h) in a suitable hydration solution at 20°C (temperatures below 20°C can lead to urea crystallization). This can be done either in a re-sweling tray or directly in a stripholder. Regardless of the method chosen, it is important to ensure that any air bubbles between the strips and the bottom of the container are removed. After this step, the strip should be covered with a non-conductive oil. If the hydration is carried out in the dissolution tray, it must also be transferred to the strip tank and run. the IEF parameter settings depend on the strip length but are always done at 20°C. If the temperature is higher than 20°C, it may result in the formation of a large number of bubbles. Temperatures above 20°C may result in protein modifications such as carbamylation due to ammoniumcyanate from urea. Once isoelectric focusing is complete, drain off the excess cover oil and store the strip at -80°C. This note assumes that the instrument used is the GE Healthcare IPGphor.
Strip Hydration
( 1 ) For 24 cm IPG tape: Melt 4 ml of hydration solution and add DTT and IPG ampholyte to the following concentrations: DTT 40 mmol/L; ampholyte 0.5% (V/V).
( 2 ) For IEF of pH 3~10, add appropriate amount of sample to the hydration solution to sample 400 μg of protein per strip. Ideally, 50-100 μl of sample per strip should be used. For 24 cm strips, the total volume of protein sample and hydration solution should be 450 μl. Ideally, the ratio of hydration solution to sample should not be less than 3:1.
( 3 ) Whether hydration is performed in the IPG Manifold or directly in a single strip, add 450 μl of buffer and protein mixture by pipetting from the end of the 24 cm, 12 lane reswelling tray (see Note 4).
( 4 ) For IEFs between pH 6 and 11, the total volume of protein and buffer used is the same as that for pH 3 to 10, except that the amphoteric electrolyte is pH 6 to 11 instead of pH 3 to 10. In addition, the hydration solution is added from one end of the tank and the sample is added separately at the positive end (+ ).
( 5 ) Place the ImmobilineDryStrip, adhesive side down, in the trough of the lysis tray, making sure that there are no air bubbles under the strip.
( 6 ) Cover with DryStrip covered oil, seal with lid and hydrate at 20°C for 16~18 h ( overnight ).
( 7 ) Remove the adhesive strip (using tweezers) and remove the excess covering oil.
( 8 ) Place glue side up in a 24 cm IPGPhor Manifold Multi-Purpose Tray (up to 12 strips).
( 9 ) Arrange the strips in the center of each slot and cover with DryStrip Covering Oil. Fill all slots with covering oil, even if not in use.
( 10 ) Dampen the electrode filter sheets to remove excess moisture.
( 11 ) Place the electrode filter sheet on each end of the adhesive strip, lightly touching the strip.
( 12 ) Attach the removable electrode to the 24 cm IPGPhor Manifold Multifunction Tray. Place the electrode on the portion of the filter paper sheet that is in contact with the adhesive strip.
( 13 ) Secure the electrodes in place and close the lid of the IPGPhor Electrophoresis Instrument.
( 14 ) The conditions for the IEF run must be determined empirically for different extracts. However, the following parameters apply to flour/Tris-CaCl2 extracts: 500 V, 1 h; 1000 V, 1 h; 8000 V, 8.20 h. Total 65,500 Vh.
( 15 ) At the end of the IEF, remove the strips, remove the excess covering oil, and place the strips in 10 ml plastic pipettes (remove the tips to allow the strips to fit). Seal with sealing film and store at -80°C.
3.3 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
3.3.1 The GE Healthcare Ettan Dalt II was used.
( 1 ) Glass plates were soaked overnight in detergent, rinsed in deionized water, then rinsed in ethanol and RO (reverse osmosis) water, and air dried.
( 2 ) Assemble the gel plate according to the instructions for use (see Note 5 on the use of silicone sealant, if using a non-self-sealing gel plate, make sure that the plate is completely sealed around the perimeter).
( 3 ) When preparing multiple pieces of gel, load the gel cassettes into the gel-making cassette by separating each cassette with a reusable plastic spacer. Ensure that the gel cassettes are tightly filled and that the front panel forms an intact seal with the gel-filling cassette by applying a rubber gasket with silicone grease. Tighten the bolts on the panel and place the gel box horizontally.
( 4 ) Attach the gel hose and funnel, and keep it vertical with the clamp and stand.
( 5 ) If using Replacement Fluid, add 100 ml of Replacement Fluid to the Equilibrium Chamber when the funnel is ready. If Replacement Solution is not used, polymerized acrylamide can be flushed from the tube with plenty of hot water after gluing.
(6) For 12 10% gels use 1 L of gel solution: 250 ml 1.5 mol/L Tris-HCl, pH 8.8; 333 ml 30% (V/V) Duracryl 0.65% (m/V) bis; 407 ml water. Stir and degas for 15 min in a 2 L Brillouin flask.
( 7 ) Add 10 ml 10% (m/V) SDS with slight stirring.
( 8 ) Add 2.5 ml 10% APS and 0.5 ml TEMED just before use and stir.
( 9 ) Fill the funnel with the acrylamide solution and keep topping it up (it is important not to empty the funnel completely of solution or bubbles will form in the gel). Keep adding solution until the gel surface is 1~2 cm ( about 1 L ) from the top of the first front plate.
( 10 ) Remove the funnel and allow the replacement solution to flow down into the V-groove at the bottom of the caster.
( 11 ) Cover the top of each piece of gel with 1 ml of water-saturated n-butanol and leave it for 3-4 h to allow the gel to polymerize.
( 12 ) - Once polymerization is complete, dismantle the caster, remove the gel cartridge, and rinse the cartridge with deionized water to remove the water-saturated butanol, as well as any small pieces of gel that may have adhered to the outside of the cartridge.
( 13 ) Prepare a 1 : 4 (RO water) dilution of 1.5 mol/L Tris-HCl (pH 8.8) for use as a gel storage solution.
( 14 ) Place the gel into a large plastic box and add enough gel storage solution to completely cover the gel.
( 15 ) The gel is ready for use after 10 to 14 days of "maturation" at 2 to 4°C, but cannot be used after one month from the date of gel preparation.
3.3.2 Gel electrophoresis
( 1 ) Thaw each IPG strip overnight in a refrigerator or cold room at 4℃ with 10 ml of frozen pre-made equilibrium solution.
( 2 ) Prepare 7 L of lower tank solution: 25 mmol/L Trizma base, 15 mmol/L glacial acetic acid.
( 3 ) Pour the buffer into the Ettan Dalt ll tank and turn on the pump to cool to 15°C.
( 4 ) Prepare the upper tank: 3 L of 200 mmol/L Tris base, 0.4% (m/V) SDS, 200 mmol/L Tricine.
( 5 ) Rinse the IPG tape with water to remove excess covering oil and equilibrate in 10 ml of equilibrium solution containing 1% (m/V) DTT for 15 min.
( 6 ) Discard the equilibrium solution and add 10 ml of equilibrium solution containing 4% (m/V) iodoacetamide and incubate for 15 min. Since iodoacetamide is photosensitive, the tubes should be wrapped with aluminum foil.
( 7 ) Remove the second gel from the refrigerator and wash the top of the gel 3 or 4 times with the upper tank solution.
( 8 ) Fill the gel cassette tank with top tank solution and load the gel strip. By convention, the acid end of the strip is on the left side of the gel. Make sure there are no air bubbles between the strip and the gel.
( 9 ) Dampen the edges of the gel cartridge to allow smooth passage through the rubber sealing tube and place the gel into the electrophoresis tank. If there are less than 12 gels, use a Perspex blank.
( 10 ) Add top tank solution to avoid disturbing the gel cassette tank, close the lid, and run with the following parameters: Step 1: Constant Power = 60 W ( 5 W/gel); Time 0.15 h; Temperature = 15°C; Pump = Automatic. Step 2: Constant power = 120 W ( 10 W/gel); time 2 h; constant power = 240 W ( 20 W/gel); time 6 h; temperature = 15°C; pump = automatic.
( 11 ) Stop electrophoresis when the dye front edge is 0.5~1.0 cm from the bottom of the gel. The total running time was about 8 h.
( 12 ) Pry open the glass case and cut the corner of each gel to indicate the direction. Place the gel into the appropriate dye solution (see later).
3.4 Gel Staining
In order to study protein spots on 2D gels by proteomics, the staining must be compatible with mass spectrometry and as sensitive as possible. Several staining methods are available that meet these criteria, including colloidal kojic acid stains, some silver stains, and a variety of fluorescent stains such as Sypro Ruby, Orange, Deep Purple, and Flamingo. colloidal kojic acid stains are not as sensitive as silver and fluorescent dyes, but are easy to use and relatively inexpensive (see the section on sensitivity in Note 6). Silver staining is a multi-step process, but can visualize lower abundance proteins. However, since there is no termination point, highly abundant proteins may be overstained. Also, the reproducibility of silver staining can be poor because the degree of staining depends on the operator. All staining procedures need to be performed on an oscillator or shaker to provide gentle oscillation during the staining/decolorization process.
3.4.1 Colloidal Caulmers Brilliant Blue G250
( 1 ) Fix the gel overnight in 50% (V/V) methanol, 10% (V/V) acetic acid (or TCA).
( 2 ) Stain in colloidal Kaumas dye (diluted with methanol according to instructions) (Bio- rad; 4 : 1 dye: methanol).
( 3 ) Decolorize with 25% (V/V) methanol, 7% (V/V) acetic acid for 1~5 min.
( 4 ) Decolorize with 25% (V/V) methanol overnight and store in water.
3.4.2 Silver staining (refer to [ 13 ])
( 1 ) Fix the gel with 40% (V/V) ethanol, 10% (V/V) acetic acid (or TCA) for 30 min.
( 2 ) Place in "sensitizing solution" for 30 min: 30% (V/V) ethanol, 0.2% (m/V) sodium thiosulphate, 0.5 mol/L sodium acetate.
( 3 ) Wash three times with distilled water for 5 min each time.
( 4 ) Add silver solution [ 0.25% (m/V) silver nitrate ] and stain for 20 min.
( 5 ) Wash twice with distilled water for 1 min each time.
( 6 ) Place in "Developing Solution": 0.24 mol/L sodium carbonate, add 200 μl of 37% formaldehyde (0.0074% final concentration) per liter of solution.
( 7 ) Add fresh developer after the solution becomes cloudy or after 10 minutes. Continue developing until the desired degree of staining is achieved.
( 8 ) Terminate the reaction by adding 0.04 mol/L EDTA for 10 min.
( 9 ) Wash twice with distilled water.
( 10 ) Stored in water.
3.4.3 Sypro Ruby Protein Staining
( 1 ) Gel was fixed with 40% (V/V) methanol, 10% (V/V) TCA overnight (18 h ).
( 2 ) Stain with Sypro Ruby dye in the dark overnight.Sypro Ruby is light sensitive, so the gel needs to be handled in a dark room and the gel storage box covered with aluminum foil.
( 3 ) The gel was decolorized with 10% (V/V) methanol, 6% (V/V) TCA.
(See Note 7 on the use of Sypro Ruby and disposal of waste liquids).
3.5 Gel imaging and analysis
Imaging systems are available from various manufacturers. The scanner must be of high resolution and, if gel spot mass spectrometry is required, the scanner must be combined with a spot picker.
Similarly, a number of software packages are available that allow gel matching to produce synthetic (or reference) gels. Spot volumes, peak heights and peak areas can also be obtained from different packages, allowing comparison of treatments. Further data analysis, such as Principal Component Analysis (PCA), is necessary, so data from gel analysis packages must be convertible to Excel spreadsheets and compatible with statistical packages.
3.6 Decolourization of the gel block prior to enzymatic digestion (cf. [ 14 ])
3.6.1 For Caulmers and Sypro Ruby Stained Gel Blocks
( 1 ) Soak in water for 15 min, discard the water and continue to soak in 50:50 water:acetonitrile for another 15 min.
( 2 ) Repeat the above washing procedure.
( 3 ) Pour off all liquid and cover the block with acetonitrile for 5 min, at which time the block should be opaque and dehydrated.
( 4 ) Discard the acetonitrile, add 0.1 mol/L NH4CO3 to rehydrate the gelatin block, and leave for 5 min.
( 5 ) Add acetonitrile to make the ratio of acetonitrile: NH4CO3 1:1 and incubate for 15 min.
( 6 ) Discard the liquid, centrifuge under vacuum to dry.
3.6.2 For silver-stained gelatine block
( 1 ) Place the block in Farmer's Reducing Agent [20% (m/V) sodium thiosulfate, 1% (m/V) potassium ferricyanide mixed with water 1 : 1 : 1] until the block is completely clean. Rinse with water.
( 2 ) Discard the liquid and dry by centrifugation under vacuum.
3.7 Reduction and alkylation (refer to [ 14 ])
( 1 ) Soak the colloid in 10 mmol/L DTT, 0.1 mol/L NH4HCO3 at 56°C for 45 min to swell. The excess liquid was poured off and quickly replaced by 55 mmol/L iodoacetamide, 0.1 mol/L NH4HCO3, and incubated for 30 min at room temperature in the dark.
( 2 ) Discard the iodoacetamide solution and wash the pellet with 1:1 acetonitrile:0.1 mol/L NH4HCO3 for 15 min.
( 3 ) Discard the liquid, centrifuge under vacuum to dry.
3.8 Enzymatic digestion
Tryptic in-gel digestion (refer to [ 14 ]) : The
( 1 ) Add sufficient amount of trypsin solution to just cover the gelatin block and ice bath for 45 min to swell it.
( 2 ) Discard the excess liquid and add 25 mmol/L NH4HCO3/5 mmol/L CaCl2, just covering the gelatin block.
( 3 ) Incubate at 37℃ overnight.
( 4 ) Peptide isolation: Collect the digested material at the bottom by centrifugation, add 25 mmol/L NH4HCO3 in the smallest volume and incubate for 15 min at room temperature.
( 5 ) Add equal volume of acetonitrile, mix well and incubate for 15 min at room temperature.
( 6 ) Collect the supernatant and repeat the extraction twice with 5% (V/V) formic acid: acetonitrile (1 : 1).
( 7 ) Combine the supernatants and centrifuge under vacuum to dry.
3.9 Peptide Desalting and Concentration for Mass Spectrometry Using Zip Tips
3.9.1 MALDI - MS
( 1 ) 10 μl of 0.1% (V/V) TFA resuspended digest.
( 2 ) 10 μl 50: 50 acetonitrile: water to wet the pipette tip and repeat twice.
( 3 ) 10 μl 0.1% (V/V) TFA equilibrated pipette tip and repeated twice.
( 4 ) Pipette up and down 10 times to draw the sample into the tip (adsorb the peptide to the tip).
( 5 ) Wash the tip with 0.1% TFA.
( 6 ) Elute with 4 μl 50:50 acetonitrile:water, 0.1% TFA.
3.9.2 ESI-MS ( see [ 15 ] )
( 1 ) 10 μl of 4% (V/V) methanol resuspended digest containing 1% (V/V) formic acid.
( 2 ) 10 μl of 1% formic acid to wet the pipette tip and repeat twice.
( 3 ) Pipette up and down 10 times for protein digestion.
( 4 ) Wash the tip with 10 μl of 0.1% (V/V) formic acid.
( 5 ) Elute with 4 μl of 70% (V/V) methanol containing 1% (V/V) formic acid.
3.10 MALDI Target Plate Sampling
( 1 ) Mix 1 μl of digest with 1 μl of substrate.
( 2 ) Spot the sample on the MALDI target plate and air dry at room temperature.