Identification of Post-translational Modifications of Plant Protein Complexes

Note: all steps are done at room temperature, unless stated otherwise.

1. Preparation of Plant Materials and Buffers

1. For N. benthamiana
   1. Grow the Agrobacterium tumefaciens strains of interest for transient expression (in this example Prf-FLAG, Prf-3xHA, Pto-FLAG and empty vector as a control) by shaking (200 rpm) in liquid culture (L medium with appropriate antibiotics) at 28 °C until stationary phase.
   2. Collect and pellet agrobacteria by centrifugation (3,000 x g for 5 min). Discard supernatant and resuspend pelleted agrobacteria in infiltration buffer.
   3. Measure the amount of agrobacteria by obtaining the optical density (OD) value at an absorbance of 600 nm (Abs 600 nm). Adjust the OD of bacteria to 0.1-0.8.
   4. Infiltrate 4 week old N. benthamiana plants (22 °C, 16 hr light) with agrobacteria by hand (with a 1-ml needleless syringe) or by vacuum (add 0.02% v/v surfactant).
      Note: Use the first nearly-fully expanded leaf, and the two immediately older leaves for most effective protein expression. For detection of ubiquitinated or acetylated proteins infiltrate leaves with 100 nM MG-132 or 100 ng/ml Trichostatin A, respectively, at 1 and 2 days post-infiltration.
   5. Harvest the infiltrated leaves 2-5 days post-infiltration and freeze in liquid nitrogen. Store the samples in a -80 °C freezer.
      Note: Determine the best level of expression of the protein of interest beforehand by taking samples over a time course of 5 days and detect protein levels by immunoblotting.
2. For A. thaliana stable transgenic lines
   1. Grow A. thaliana seeds in 6-well plates supplemented with 5 ml of liquid MS medium (1% w/v sucrose) per well. Put three to five seeds (sterilized and stratified) of the transgenic line of interest or the untransformed control line per well. Shake at 200 rpm for two days before transferring the plate to a growth room and leave for 2 weeks (22 °C, 10 hr light).
   2. Harvest samples and freeze in liquid nitrogen.
      Note: You can use as a control a transgenic line expressing an unrelated protein with the same tag as the protein of interest.
3. Buffers
   1. Prepare Buffer A. De-gas the buffer for 1-2 hr (for RLKs, other membrane bound proteins and nuclear proteins, add 1% v/v IGEPAL CA-630⁵³).
      Note: You can keep Buffer A at 4 °C indefinitely. You can use 1% v/v Triton instead of IGEPAL CA-360.
   2. Prepare 80 ml of cold Buffer B at least 1-2 hr before extraction.
      Note: The ideal ratio of tissue vs. extraction buffer is 1:4 (w/v).

2. Protein Extraction

1. Just prior to extraction prepare 80 ml of cold Buffer C.
   Note: Add 10 μM MG-132 for the detection of ubiquitinated proteins.
2. Grind 20 g of N. benthamiana tissue (approximately 15 leaves) or A. thaliana seedlings (2-4 g per 6-well plate) in liquid nitrogen using a mortar and pestle.
3. Add 80 ml of cold Buffer C to the 20 g of ground tissue, mix well, and thaw on ice.
   Note: Grind all samples at the same time (protein of interest and control).
4. Homogenize with 3 bursts of 10 sec each at full speed with a tissue homogenizer (prechilled at 4 °C). Filter through a 22-25 μm tissue and centrifuge at 30,000 x g for 30 min at 4 °C.
   Note: Alternatively, homogenize with a fresh prechilled mortar and pestle.
5. For the blocking and washing steps of the affinity matrix, prepare 20 ml of Buffer D.
6. Block 100 ml of suitable affinity matrix using 500 ml of cold Buffer D containing 1 % BSA for 5 min at 4 °C.
   Note: Preferred affinity matrices include anti-FLAG M2 agarose, Streptavidin agarose, GFP-Trap_A and anti-HA agarose.
7. Wash 3x with 1 ml cold Buffer D.
8. Remove supernatant of leaf extract and filter through a 0.45 μm syringe filter into a new tube on ice.
   Note: The extract color should be green or yellow; if the sample has turned brown, discard and start over.
9. Take an aliquot of the crude extract for immunoblot, add 5x SDS-PAGE loading buffer, vortex and denature by boiling samples for 10 min at 80-100 °C in a heat block.

3. Protein Immunoprecipitation

1. Split the protein extract into two 50 ml tubes and add 50 μl of the affinity matrix suspended in Buffer D to each tube. Incubate with gentle rotation for 2 hr at 4 °C.
   Note: Longer incubations have not been found to increase yield.
2. Prepare 600 μl (6x bead volume of affinity matrix) of elution buffer by adding to Buffer D (final concentrations): 0.5% BSA, 0.25 mg/ml FLAG peptide (for anti-FLAG M2 agarose) or 10 mM D-biotin (for Streptavidin agarose).
   Note: Elution is not recommended in the case of GFP-Trap_A and anti-HA matrices, so proceed directly to boiling in 1x SDS-PAGE loading buffer, step 3.11.
3. Precipitate the affinity matrix by centrifugation at 4 °C (5 min at 1,000 x g).
4. Take an aliquot of the unbound extract for immunoblot, discard the rest of the supernatant, leaving approximately 500 μl in the bottom of the tube.
5. Resuspend the slurry solution with a wide-bore tip.
6. Transfer the mixed slurry solution from both tubes to a single 1.5 ml tube.
   Note: From now on, use 1.5 ml low-protein binding microfuge tubes.
7. Pulse 3x for 5 sec using a benchtop centrifuge to precipitate the affinity matrix and remove the supernatant.
8. Wash 3-5x with 1 ml cold buffer D. Between the washes precipitate the affinity matrix as previously described and discard the supernatant. In the last wash remove excess Buffer D with the needle of a syringe.
   Note: A final wash using Buffer D with 0.1% IGEPAL can be used to further reduce nonspecific binding.
9. Elute with 200 μl of the appropriate elution buffer (for anti-FLAG M2 or Streptavidin agarose see step 3.2) 3x, 5 min each time with constant shaking.
10. Pool the 3 eluates in a single tube. Concentrate the proteins by using 30 μl of absorption resin. Vortex, let stand for 5 min and precipitate the resin (2 min at 10,000 x g). Discard the supernatant.
    Note: For GFP-Trap_A and anti-HA agarose, skip the steps 3.9 and 3.10.
11. Add 50 μl of 1x SDS-PAGE loading buffer to the precipitated affinity matrix or absorption resin. Vortex and boil for 10 min at 80-100 °C in a heat block.
12. Centrifuge the boiled affinity matrix or resin for 2 min at 10,000 x g. Aliquot 5 μl of the supernatant for immunoblot, and run with other fractions onto a ~10 cm long SDS-PAGE gel (Figure 2).
13. For separation and identification of phosphorylated proteins by mass spectrometry, load the remaining 45 µl onto a ~19-cm long SDS-PAGE gel if extra separation is required (Figure 2).
    Note: A blank lane can be included between all samples to reduce contamination of samples.

4. Protein Digestion

1. Stain the SDS-PAGE gel with colloidal Coomassie Brilliant Blue (cCBB) stain.
   Note: Minimize handling of the gel to reduce contamination with keratins. Make sure that none of the equipment or tools have been used for immunoblotting or other activities that might have left residual protein contamination.
2. Destain the gel with copious washing in water, preferably O/N. Cut the band of interest from the gel with a clean razor blade.
   Note: Align the gel with the immunoblot if necessary to locate the correct area. As phosphorylation can delay migration of proteins on SDS-PAGE cut the area immediately above and below the band of interest.
3. Dice the gel slice into cubes of 2-4 mm (this ensures that the gel will be covered by solutions in the tube without blocking pipette tips). Place in a tube.
4. Estimate the volume required to keep the gel well submerged. Use this amount for derivatization, incubation with protease and peptide extraction, and then use double to triple volumes for washing.
   Note: In a typical digest with approximately 100 μl of the cut gel pieces, use 500 μl solution for washing, 2 x 180 μl for dehydration, 150 μl for reduction, 120 μl for alkylation, and 120 μl for digestion.
5. Wash the gel pieces 2 x 20 min (or until destained) by adding 50% acetonitrile (in 50 mM ammonium bicarbonate, ABC). Pipette off the solution taking care not to remove gel pieces.
6. Dehydrate with 100% acetonitrile for 5 min and remove free liquid.
   Note: The gel should appear shrunken and white. If the blue color persists, incubate longer in acetonitrile/ABC and/or at elevated temperature (45-55 °C).
7. Reduce protein disulfide bonds by adding 10 mM DTT in water and incubate for 30-45 min at 56 ºC with shaking. Remove free liquid.
8. Alkylate cysteine residues by addition of 55 mM chloroacetamide (in 50 mM ABC) for 20-30 min (room temperature, dark). Remove free liquid.
9. Wash gel pieces 2 x 10 min with 50% acetonitrile (in 50 mM ABC). Remove free liquid.
10. Dehydrate gel pieces with 100% acetonitrile for 5 min and remove free liquid.
11. For the tryptic digest, add 40 μl of trypsin working solution (100 ng of trypsin in digestion buffer: 50 mM ABC, 5% acetonitrile for an average Coomassie-stained band). Allow gel pieces to rehydrate for 10 min. Add enough of digestion buffer to cover gel pieces if necessary. Incubate at 37 ºC O/N.
12. To stop the digestion and extract the peptides from the gel pieces, add 5% formic acid (in 50% acetonitrile) (add the same volume as the digestion volume). Sonicate for 5-10 min. Transfer the supernatant to new tube. Repeat the extraction 3x.
13. Dry the peptides by lyophilization (preferred) or a vacuum concentrator (such as Speed-Vac or Savant). Store at -20 ºC.
14. Submit your sample for mass spectrometry.
    Note: We submitted our sample to the mass spectrometry facility at The Sainsbury Laboratory (Norwich, UK). Protocols at mass spectrometry facilities vary considerably, but typically peptides will be dissolved in 0.2% trifluoroacetic acid with 2% acetonitrile before loading onto a liquid chromatography system coupled to electro-spray tandem mass spectrometry.