In vitro phosphorylation assay and LC-MS/MS

In vitro phosphorylation assay:

Please be noted that this protocol is part of the paper (Wang L*, Yan X*, Li Y, Wang Z, Chhajed S, Shang B, Wang Z, Choi SW, Zhao H, Chen S, Zhang X. PRP4KA phosphorylates SERRATE for degradation via 20S proteasome to fine-tune miRNA production in Arabidopsis. Sci Adv. 2022 Mar 25;8(12):eabm8435. doi: 10.1126/sciadv.abm8435. Epub 2022 Mar 25.

PMID: 35333566). When this protocol is used, please cite the above-mentioned paper.

1. Purified proteins including the kinase (PRPK4A) and potential substrates (SE, Hyl1).
2. Mixed 1 μg Kinase protein and 3 μg substrate protein in the 1.5 mL tube with double-distilled water (ddH₂O) up to 15 μL.
3. Then, the mixed proteins were added into 15 μL of 2× phosphorylation buffer.
4. The reactions were incubated for 2-4 hours at room temperature.
5. Then, addition of 7.5 μL of 5× SDS loading buffer.
6. The samples were denatured at 95 ℃ for 5 min.
7. The 10-15 μL of samples were resolved by 10 % SDS-PAGE gel (depend on protein size) until the dye run out of the gel, followed by the detection by auto-radiography.
8. Fixed and washed the gel using the buffer A for 10 min, 3 times.
9. Dried the gel on the vacuum drier at 80 ℃ for 2 hours and 30 min.
10. Developed the signal with the film.

Buffer:

1. 2× phosphorylation buffer:

20 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 100 mM NaCl, 5 mM EDTA (pH 8.0), 1

mM dithiothreitol (DTT), 100 μM ATP,     and 0.5-2 μL of [γ-32 P ] ATP of each sample.

2. 5× SDS loading buffer:

250 mM Tris-HCl (pH 6.8), 10 % SDS, 0.5 % bromophenol blue, 50 % glycerine, 5 % 2-ME.

3. Buffer A:

10 % ethanol, 10 % Acetic Acid.

LC-MS/MS

Be noted: “the phosphoproteomics protocol reported here will be published as a part in a book chapter in Methods in Enzymology to be published in the near future.”. The citation would be as follows:

Noe, P., Tan, B., Dufresne, C.P., Chen, S. (2022) Proteomics and phosphoproteomics of C3 to CAM transition in the common ice plant. Methods in Enzymology

Gel destaining and bands excision:

1. For the MS assay, the in vitro phosphorylation assays were per formed in parallel but with cold ATP (without [γ-32 P ] ATP). The reaction was loaded onto SDS-PAGE gels.
2. After separation, rinse the gel in a plastic tray during 3 periods of 5 minutes with enough ddH₂O to cover the gel.
3. Remove the water and add Bio-Safe Coomassie stain to the plastic tray until the gel is completely covered. Put the tray on a rocking platform shaker for 1 hour.
4. Rinse the gel in ddH₂O for few hours to allow for good visualization of the bands. A single band should appear for alpha-casein. Image the gel if needed at this point.
5. Carefully excise all the bands with a clean blade and cut them into approximately 2×2 mm cubes to minimize the length of the subsequent destaining step. Transfer the excised and cut bands in a 1.5mL tube. If some proteins are stuck in the stacking gel (presence of dye in the stacking gel), it is preferable to also excise it and include it in the 1.5mL tube.

Bands destaining, reduction and alkylation:

6. Add 300 µL 100% acetonitrile (ACN) to the gel pieces and incubate during 10 minutes on shaker or vortex mixer. Spin down and remove ACN.

7. Add 300 µL 100 mM ABC/ACN (1:1 V/V). Incubate on shaker for 30 minutes. Spin down and remove all liquid from the tube.

8. Repeat steps 6 and 7 until the blue stain from the Coomassie Brilliant Blue (CBB) has been completely removed. Shaking the tubes containing the gel pieces on a thermomixer at 50°C can help to destain faster. It is possible to store the gel pieces in the 100 mM ABC/ACN solution overnight at 4°C at this stage.

9. Add 300 µL 100% ACN. Incubate for 5 minutes and remove all liquid from the tube.

10. Allow for the gel pieces to dry for several minutes. It is also possible to dry the samples in a SpeedVac for one minute.

11. Add enough 10 mM dithiothreitol (DTT) in 100 mM Ammonium Bicarbonate (ABC) to cover the gel. Incubate on a thermomixer at 55°C and 700 revolutions per minute (rpm) for 45 minutes, and remove all liquid.

12. Add enough 55 mM fresh chloroacetamide in 100 mM ABC to cover the gel. Incubate at 60 minutes in the dark at room temperature for alkylation. Remove all liquid from the tube.

13. Wash the gel pieces with 300 µL 20 mM ABC/ACN (1:1 V/V). Incubate at room temperature for 15 min. Remove all liquid from the tube.

14. Dry the gel pieces in a SpeedVac for 3 minutes.

Trypsin digestion:

15. Add trypsin with an enzyme to protein ratio of 1:80 (w/w). Add enough 50 mM ABC to cover the gel pieces. Incubate at 37 °C for 30 min. If the gel pieces absorbed some of the liquid, add more 50 mM ABC to cover the gel pieces.

16. Incubate at 37°C for overnight.

17. Centrifuge and transfer all liquid a new 1.5mL tube. Add 30 µL 80% ACN with 20% of 1% formic acid (FA) to the gel pieces to stop the reaction. Incubate on a shaker at room temperature for 30 minutes.

18. Centrifuge and transfer all liquid to a new 1.5 mL tube.

19. Add 30 µL 60% ACN to the gel pieces. Vortex for 10 min, and combine all liquid with the previous peptide extract (from step 17).

20. Dry the sample using a SpeedVac (typically for 1 hour). At this stage, the samples can be stored at -20 °C for few months until phosphoenrichment.

Phosphoenrichment:

1. Resuspend the digested peptides in 25 µL 0.1% FA water and 25 µL load buffer.
2. Sonicate for 3 minutes, and vortex during 30 minutes.
3. Equilibrate the TiO2 + ZrO2 NuTip™ by pipetting 20 µL of load buffer and releasing into a waste beaker. Repeat this step 20 times.
4. Take a new 1.5 mL tube and label it “flow-through”.
5. Load 25 µL of the peptide mixture into the TiO2 + ZrO2 NuTip™. Slowly pipet up and down for 10 minutes into the “flow-through” tube to allow for specific binding of the phosphopeptides to the embedded chromatographic material. It is crucial to perform this step very carefully, as it will determine the efficiency of the phosphoenrichment process. It is also important to avoid the formation of air bubbles to ensure a constant flow of digested peptide solution through the tip media. After 10 minutes, release all liquid into the “flow-through” tube.
6. Load 25 µL of the wash buffer into the TiO2 + ZrO2 NuTip™ and transfer it to the “flow-through” tube.
7. Load 25 more µL of the wash buffer into the TiO2 + ZrO2 NuTip™. Pipet up and down 20 times and release all liquid into the “flow-through” tube.
8. Take a new 1.5 mL tube and label it “eluate”.
9. Load 7 µL of the elution buffer into the TiO2 + ZrO2 NuTip™. Pipet up and down 10 times into the “eluate” tube. Repeat this step one time by pipetting up and down in the cap of the “eluate” tube. Combine both solutions.
10. Re-equilibrate the TiO2 + ZrO2 NuTip™ by pipetting 20 µL of load buffer and releasing into a waste beaker. Repeat this step 25 times.
11. Load the remaining 25 µL of the peptide mixture into the TiO2 + ZrO2 NuTip™. Slowly pipet up and down for 10 minutes. After 10 minutes, release all liquid into the “flow-through” tube.
12. Load 25 µL of the wash buffer into the TiO2 + ZrO2 NuTip™ and transfer it to the “flow-through” tube.
13. Load 25 more µL of the wash buffer into the TiO2 + ZrO2 NuTip™. Pipet up and down 20 times in the cap of the “flow-through” tube. Combine the liquid with the remaining solution in the “flow-through” tube.
14. Load 7 µL of the elution buffer into the TiO2 + ZrO2 NuTip™. Pipet up and down 10 times into the cap of the “eluate” tube and combine the liquid with the remaining solution in the “eluate” tube.
15. Repeat step 14 one additional time.
16. To avoid loss of the phosphate group under highly basic condition, the eluted peptides were acidified with 5 µL 100% formic acid.
17. Dry down the enriched samples and the flow-through in a SpeedVac. The dried samples can be kept for few months at -20 °C before use.

Sample processing by LC-MS/MS:

1. For each biological sample to be analyzed, two peptide samples are introduced into the LC-MS/MS: the phosphoenriched sample and the total protein sample for a qualitative and quantitative proteome analysis.
2. In addition, the sample corresponding to the flow-through of the enrichment step can be analyzed for troubleshooting purposes.
3. Before starting the MS analysis, the phosphoenriched and the total protein samples need to be resuspended in 13 µL 0.1% formic acid solution (V/V) for injection of 10 µL on the LC. Sonicate the samples for 5 min, vortex during 30 minutes, and centrifuge at 14000 rpm for 15 min. Below is the instrument method used for the samples presented in the next section.
4. Peptides were directly introduced into a Vanquish Neo ultra-high-performance liquid chromatography (UHPLC) (Thermo Scientific) connected to an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific).
5. Peptides were injected through direct mode with loop offline at a loading flow of 100 µL/minute at a pressure of 1500 bar. Similarly, fast column equilibration was performed at a flow of 100 µL/min and at a pressure of 1500 bar to reduce excessive computation time.
6. The peptide samples were loaded onto an Acclaim PepMap 100 C18 precolumn (20 mm by 75 µm; 3 µm) and separated on a PepMap RSLC C18 analytical column (250 mm by 75 µm; 2 µm) at a flow rate at 300 nl/min through a linear gradient from solvent A [0.1% formic acid (v/v)] to 35% solvent B (0.1% formic acid and 99.9% ACN) in 110 min and to 98% solvent B for additional 10 min.
7. The mass spectrometer used was an Orbitrap Fusion Lumos Tribrid system with a collision-induced dissociation (CID) and electron transfer dissociation (ETD) decision tree method.
8. The Orbitrap MS1 scan range was 350 to 1800 mass/charge ratio, the automatic gain control target was set to 400,000, and the maximum inject time was set to 50 ms.
9. The MS/MS spectra were acquired in the linear ion trap after CID for two to four charges and/or ETD for three to eight charges.
10. The threshold for precursor ion selection was 5000 counts, and selected parent ions were isolated using a mass window of 1.3.

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