To identify and quantify phosphoproteins in large-format 2-D gels, it is important to prepare a high-quality protein sample that is free from interfering compounds such as carbohydrates and nucleic acids. Phenol–ammonium acetate/methanol method has been successfully used to extract proteins from developing seeds (rich in oil and carbohydrate) of soybean and rapeseed suitable for generating high-resolution 2-D gel reference maps. The phenolbased extraction method has also been used by many other labs for large-scale phosphoprotein analysis. Based on these and other gel-based proteomics studies, it can be stated that the phenol–ammonium acetate/methanol method provides high-quality protein suitable for performing high-resolution 2-DGE while minimizing proteolysis and blocking endogenous phosphatase activity during protein isolation. Nevertheless, we cannot rule out the possibility of very low levels of endogenous phosphatase or protease activity. Taking this into account, it is always good to add phosphatase and protease inhibitors.
Isolated proteins are then separated by 2-DGE. Both biological and experimental replications are required for downstream 2-D gel image analysis and quantification. The 2-DGE technique has been combined with the modified method of Pro-Q DPS to detect and quantify phosphoproteins on a large scale.
Upon image analysis of 2-D gels and expression profiling, excised 2-D spots are in-gel digested with trypsin. Extracted peptides are then subjected to MS and database analysis for identification of phosphoproteins. We have attempted to provide step-by-step information in this chapter. However, it is difficult to provide details on all the steps involved in this analysis. For example, it is almost impossible to describe the steps involved in the image analysis of 2-D gels using ImageMaster 2D Platinum software. In such cases, readers are encouraged to utilize the user manual for the ImageMaster software. We should emphasize that the detection of all distinct spots and manual editing of those spots are important steps in the 2-D gel image analysis. Recently, ImageMaster software has been updated and version 6 of this software is now being used.
1 Total Protein Extraction: Phenol– Ammonium Acetate/Methanol Method
a) Grind 250 mg of plant materials with liquid nitrogen in a mortar and pestle to obtain a powder.
b) Add 10 mL of phenol extraction buffer and continue grinding for an additional 30 s.
c) Transfer to a 15-mL Falcon tube and agitate on a nutator for 30 min at 4°C.
d) Centrifuge for 30 min at 2,800 g (4°C).
e) Transfer the upper-phase solution to a fresh 50-mL Falcon tube.
f) Add 5 volumes of ammonium acetate/methanol solution (ice cold), vortex, and incubate at −20°C overnight to precipitate the phenol-extracted proteins.
g) Centrifuge for 30 min at 2,800 g (4°C) to collect the precipitate.
h) Wash the pellet twice with ice-cold ammonium acetate/methanol solution, then twice with ice-cold 80% acetone solution, and finally with cold 70% ethanol solution.
i) Dry the final pellet (after removing the wash solution) for 20 min at 37°C.
j) Resuspend the final pellet in IEF extraction solution.
k) Centrifuge for 15 min at 28,000 g at RT.
l) Carefully transfer clear supernatant to a new 1.5-mL microfuge tube. Store in single-use aliquots at −80°C.
2 Protein Quantification: A Modified Bradford Method
a) Dilute the protein sample with 0.5× SDS running buffer in a 1.5-mL microfuge tube, and mix.
b) Use 0.5× SDS running buffer as blank and add 6 μL to the first column of the microtiter plate.
c) Add 1, 2, 4, and 6 μL of 1 mg/mL BSA standard in triplicate to wells and bring the final volume to 6 μL with 0.5× SDS running buffer.
d) Add 1 μL of tenfold diluted protein sample in triplicate to wells plus 5 μL of 0.5× SDS running buffer to make a total volume of 6 μL.
e) Add 200 μL of diluted Bradford dye to wells with protein.
f) Mix and incubate at RT for 5 min.
g) Vortex the microtiter plate on the spectrophotometer and measure absorbance at 595 nm.
h) Calculate the protein concentration.
3 Isoelectric Focusing of Total Proteins
a) Add 1 mg protein to a 1.5-mL microfuge tube and bring the final volume to 450 μL with IEF extraction buffer.
b) Add 2.25 μL (final concentration of 0.5%) of the correct IPG buffer to the same tube.
c) Mix the sample by vortexing and centrifuging at 28,000 g for 5 min to remove insoluble materials.
d) Pipette the supernatant into the IPG focusing tray starting from one end to the other.
e) Peel apart a dehydrated IPG strip (pH 4–7; 24 cm) using forceps and place the dried acrylamide side face down into the sample well in the IPG focusing tray.
f) Cover the focusing tray with the lid and keep inside a plastic bag to minimize evaporation.
g) Allow the IPG strip to rehydrate for 90 min at RT.
h) Overlay the IPG strip with mineral oil (∼2.5 mL) to prevent dehydration.
i) Place the IPG focusing tray into Protean IEF cell unit.
j) Perform active rehydration (10 h at 50 V), followed by threestep focusing protocol: 100 V for 100 V h, 500 V for 500 V h, and 8,000 V for 99 kV h.
4 Assembling the Ettan DALTtwelve Gel Caster Unit
a) Tilt the DALTtwelve unit back so that it rests on its support legs.
b) Place a thicker separator sheet against the back wall to easily remove the last cassette from the gel caster unit after polymerization.
c) Clean each glass plate carefully with deionized water and ethanol using Kimwipes.
d) Fill the caster by alternating cassettes with separator sheets. End with a separator sheet, and then use the thicker separator sheets to bring the level of the stack even with the edge of the caster.
e) Lubricate the foam gasket with a small amount of GelSeal and place it in the groove on the faceplate.
f) Turn four black-knobbed screws into the four threaded holes across the bottom until they are well engaged. Usually two to three full turns are enough.
g) Place the faceplate carefully onto the caster with the bottom slots resting on their respective screws. Screw the four remaining black-knobbed screws into the holes at the sides of the faceplate and tighten all eight evenly. The assembled unit is now ready to cast gels.
5 Casting 12% SDS-PAGE into the Ettan DALTtwelve Gel Cas
a) To cast twelve gels, add 300 mL of acrylamide stock, 188 mL of 1.5 M Tris–HCl, pH 8.8, 7.5 mL of 10% SDS, and 252 mL of deionized water in 1-L sidearm flask. This is the separating gel solution.
b) Place the flask on stir plate, add a medium size stir bar, and stir the solution.
c) Connect sidearm to vacuum, cover top opening with solid rubber stopper, and apply vacuum for 30 min.
d) Turn off vacuum, remove rubber stopper, and disconnect hose.
e) Add 3.6 mL of 10% ammonium persulfate while stirring the solution.
f) Add 120 μL of TEMED and continue to stir for 30 s. Move quickly to cast the gels.
g) Slowly pour the gel solution into the caster through the hydrostatic balance chamber until it is about 2 cm below the desired gel height.
h) Pour the displacement solution into the chamber until it is 0.25 cm below the surface of glass plates, and then immediately place the feed tube into the grommet to stop the flow.
i) Very slowly overlay each gel with 1.5 mL of deionized water.
j) Cover the upper portion of the unit with plastic wrap to prevent dehydration.
k) Allow polymerization for 16 h.
l) Bring the unit near a sink, carefully disassemble, and scrape off access acrylamide.
m) Remove the gel cassette from the caster by pulling forward on the separator sheets, rinse the outer surface of each gel cassette with deionized water to remove any polyacrylamide particles, and place gel cassettes in a cassette rack.
n) Prepare stacking gel solution by combining 10.6 mL of acrylamide stock, 20 mL of 0.5 M Tris–HCl, pH 6.8, 0.8 mL of 10% SDS, and 48.8 mL of deionized water in a 200-mL beaker.
o) Mix thoroughly by stirring the solution on a stir plate.
p) Add 0.6 mL of 10% ammonium persulfate while stirring the solution.
q) Add 40 μL of TEMED and continue to stir for 30 s.
r) Place enough stacking gel solution on top of the separating gel using a plastic transfer pipette to give 2 cm height after polymerization.
s) Overlay each gel with 1 mL isobutanol.
t) Allow to polymerize for ∼1 h.
u) Once polymerized, pour off isobutanol and wash several times with deionized water to remove any traces of isobutanol.
v) Add 1× SDS running buffer on top of the stacking gel to prevent dehydration.
6 Reduction/Alkylation of Proteins in the IPG Strips
a) Remove the IPG strips from the IPG focusing tray by holding one end of the strip with forceps and blotting off excess mineral oil using Kimwipes.
b) Use Protean IEF system 24-cm disposable tray to place the IPG strip in a well carrying 2.5 mL reduction solution, facing the gel side up.
c) Incubate on rocking platform at medium speed for 15 min at RT.
d) Transfer the IPG strips to a new disposable tray with 2.5 mL alkylation solution in each well.
e) Incubate again on rocking platform at medium speed for 15 min at RT.
f) Transfer the IPG strips again to a new disposable tray carrying 2.5 mL 1× SDS running buffer in each well for a brief wash. The IPG strips are now ready for SDS-PAGE.
a) Remove the IPG strip from the tray by holding one end of the strip with forceps and placing the IPG strip carefully on the surface of stacking gel.
b) Place a small square of electrode wick (0.5× 0.5 cm2) containing 1–2 μL of PeppermintStick phosphoprotein standards next to the acidic end of the strip.
c) Quickly overlay the IPG strip and the electrode wick with 2–3 mL agarose overlay solution.
d) Pour about 7.5 L of 1× SDS running buffer to the lower chamber of the separation unit.
e) Once agarose is solidified, insert the gel cassette into the separation unit through the buffer seal slots flanked by rubber gaskets.
f) Pour about 2.0 L of 2× SDS running buffer until the solution reaches the marked upper level on the separation unit.
g) Run the electrophoresis unit at 2 W/gel until dye migrates off the gel.
8 Phosphoprotein Detection with Pro-Q DPS and Image Acquisition
a) Take out the gel cassettes one by one from the separation unit, open the cassette, and carefully transfer the gel to a large gel-staining tray.
b) Wash the gel twice with deionized water for 10 min each. Incubate at RT with constant shaking on an orbital shaker at a speed of 35 rpm in all steps of this modified Pro-Q DPS procedure, unless stated otherwise.
c) Decant deionized water and immerse the gel in 200 mL of fixation solution. Decant the fixation solution and repeat.
d) Immerse the gel in 250 mL of washing solution for 15 min. Decant wash solution and repeat.
e) Incubate the gel in 150 mL of staining solution in the dark for 2 h and decant solution.
f) Immerse the gel in 250 mL of destaining solution and incubate in the dark for 30 min. Decant the destaining solution and repeat this step three more times. The total required destaining time is 2 h.
g) Wash the gel with 250 mL of deionized water in the dark for 5 min. Decant deionized water and repeat.
h) Scan the gel using a laser imager with 532-nm excitation and 580-nm bandpass emission filter (Fujifilm FLA 5000). Collect and analyze data as 100-μm resolution, 16-bit TIFF files.
i) Use Image Gauge Analysis softwareand ImageMaster 2D Platinum software version 5 or 6 to display and analyze data.
9 Total Protein Detection with Colloidal CBB
a) Once gel imaging is finished, immerse the gel in 250 mL of colloidal CBB with agitation for 16 h to detect total proteins and then destain in deionized water.
b) Scan and analyze the gel using ScanMaker 9800XL (300 dpi resolution and 16-bit grayscale pixel depth) and ImageMaster software, respectively.
c) Decant destain solution and add 250 mL of gel storage solution to store the gel at 4°C for up to a few months.
10 Phosphoprotein and Protein Spots Excision and In-Gel Digestion
a) Overlay the images of phosphoproteins and proteins as false colors using Adobe photoshop by aligning the phosphoprotein and protein markers.
b) Manually excise the desired phosphoprotein spots using protein spots as landmarks with the help of 0.15-mm spot picker and transfer to a 96-well MultiScreen plate.
c) Use a vacuum manifold system to process in-gel digestion reactions.
d) Destain gel plugs with 200 μL in-gel wash solution for 15 min at RT with gentle agitation on a microplate shaker at a speed of 50 rpm. Evacuate the solution from the bottom of the filter plate using a vacuum manifold system. Repeat this step two more times or until all stain is removed.
e) Dehydrate the gel plugs with 100 μL of acetonitrile for 5 min and remove acetonitrile by vacuum evacuation.
f) Remove residual acetonitrile by blotting the plate gently with Kimwipes.
g) Place a 96-well V-bottom sample collection plate underneath the MultiScreen plate.
h) Add 50 μL of in-gel trypsin solution to wells to rehydrate the gel plugs, cover the plate with adhesive film, and place the cassette inside a plastic sealable bag.
i) Incubate at 37°C for 16 h.
j) Add 100 μL of in-gel extraction solution to wells and incubate for 10 min at RT with gentle agitation.
k) Centrifuge at 2,000 × g for 2 min to collect trypsin-digested peptides into V-bottom polypropylene collection plate.
l) Repeat steps 10 and 11 once more.
m) Dry the pooled extracted peptides using CentriVap Console and store at −80°C.
11 Mass Spectrometry and Data Analyses
a) Resuspend the dried pellet in 50 μL of 0.1% formic acid.
b) Load 15 μL for mass spectral analysis on an LTQ ProteomeX linear ion trap LC-MS/MS instrument following standard procedures.
c) Search MS/MS data against the suitable database using the SEQUEST algorithm as part of the BioWorks 3.2 software suite.
d) Assign search parameters for the database and then assign protein assignment criteria to identify phosphoproteins.
- de Graauw, M. (2009). Phospho-Proteomics. Humana Press.