Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Podsumowanie

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Streszczenie

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (> 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (< 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Wprowadzenie

High salinity, low temperature, and drought cause osmotic stresses, which is a major environmental factor that affects plant productivity^1,2. Protein phosphorylation is one of the most significant post-translational modifications mediating signal perception and transduction in plant response to osmotic stress^3,4,5. SNF1-related protein kinase 2s (SnRK2s) are involved in the osmotic stress signaling⁶. Nine of ten members of the SnRK2 family show significant activation in response to osmotic stress^7,8. The snrk2.1/2/3/4/5/6/7/8/9/10 decuple (snrk2-dec) mutant having mutations in all ten SnRK2 displayed hypersensitivity to osmotic stress. In snrk2-dec mutant, the osmotic stress-induced accumulation of inositol 1,4,5-trisphosphate (IP₃), abscisic acid (ABA) biosynthesis, and gene expressions are strongly reduced, highlighting the vital role of SnRK2s in osmotic stress responses⁶. However, it is still unclear how SnRK2s kinases regulate these biological processes. Profiling the phosphoproteomic changes in response to osmotic stress is an efficient way to bridge this gap and to delineate the osmotic stress-triggered defense mechanisms in plants.

Mass spectrometry (MS) is a powerful technique for mapping plant phosphoproteome⁹. Characterization of plant phosphoproteomics, however, remain a challenge due to the dynamic range of plant proteome and the complexity of plant lysate⁴. To overcome these challenges, we developed a universal plant phosphoproteomic workflow, which eliminates unwanted interferences such as from photosynthetic pigments and secondary metabolites, and enabling the deep coverage of plant phosphoproteome¹⁰. Several phosphopeptide enrichment methods such as immobilized metal ion affinity chromatography (IMAC) and metal oxide chromatography (MOC) have been developed for enriching phosphopeptides prior to MS analysis^{11,12,13,14,15,16}. Acidic non-phosphopeptides co-purifying with phosphopeptides are the major interferences for phosphopeptide detection. Previously, we standardized the pH value and organic acid concentration of IMAC loading buffer to eliminate the binding of non-phosphopeptides, to obtain more than 90% enrichment specificity bypassing the pre-fractionation step¹¹.

Sample loss in the multi-step process of phosphopeptide enrichment and fractionation hampers the sensitivity of phosphopeptide identification and the depth of phosphoproteomic coverage. Stop-and-go-extraction tips (stage tips) are pipette tips that contain small disks to cap the end of the tip, which can be incorporated with chromatography for peptide fractionation and cleaning¹⁷. Sample loss during the stage tip procedure can be minimized by avoiding sample transfer between the tubes. We have successfully implemented stage tip in Ga³⁺-IMAC and Fe³⁺-IMAC to separate low abundant multiple phosphorylated peptides from singly phosphorylated peptides, which improved the depth of human phosphoproteome¹⁵. In addition, the use of high pH reversed-phase (Hp-RP) stage tip has demonstrated the wider coverage of human membrane proteome compared to that of strong cation exchange (SCX) and strong anion exchange (SAX) chromatography¹⁸. Therefore, integrating IMAC and Hp-RP stage tip techniques can increase plant phosphoproteome coverage with simplicity, high specificity, and high throughput. We have demonstrated that this strategy identified more than 20,000 phosphorylation sites from Arabidopsis seedlings, representing an enhanced depth of plant phosphoproteome¹⁹.

Here, we report a stage tip-based phosphoproteomic protocol for phosphoproteomic profiling in Arabidopsis. This workflow was applied to study the phosphoproteomic perturbation of wild-type and snrk2-dec mutant seedlings in response to osmotic stress. The phosphoproteomic analysis revealed the phosphorylation sites implicated in kinase activation and early osmotic stress signaling. Comparative analysis of wild-type and snrk2-dec mutant phosphoproteome data leaded the discovery of a Raf-like kinase (RAF)-SnRK2 kinase cascade which plays a key role in osmore stress signaling in high plants.

Protokół

1. Sample preparation

1. Harvest the control and stress treated seedlings (1 g) in an aluminum foil and flash freeze the samples in liquid nitrogen.
   NOTE: Higher protein concentration is usually observed from two-week-old seedlings than that from mature plants. One gram of seedlings generates approximately 10 mg of protein lysate, which is enough for the MS analysis. All centrifugation steps take place at 16,000 x g in step 1.
2. Grind frozen seedlings into a fine powder using a mortar and pestle filled with liquid nitrogen.
3. Add 1 mL of lysis buffer ((6 M guanidine-HCl in 100 mM Tris-HCl, pH 8.5) with 10 mM Tris (2-carboxyethyl)phosphine hydrochloride (TCEP), 40 mM 2-chloroacetamide (CAA), protease inhibitor and phosphatase inhibitor cocktails) to the mortar and mix with the aid of pestle.
4. Transfer the plant lysate to a 1.5 mL tube and heat at 95 °C for 5 min.
5. Place the tube on ice for 10 min. Sonicate the tube on ice for 10 s, pause for 10 s, and repeat thrice.
6. Centrifuge the tube for 20 min and aliquot 150 µL of plant lysate into a 1.5 mL tube.
7. Add 600 µL of 100% methanol into the tube. Vortex and spin down the tube.
8. Add 150 µL of 100% chloroform into the tube. Vortex and spin down the tube.
9. Add 450 µL of ddH₂O into the tube. Vortex and centrifuge the tube for 3 min.
10. Discard the upper aqueous layer. Add 600 µL of 100% methanol into the tube. Centrifuge the tube for 3 min and then discard the solution.
11. Wash protein pellets with 600 µL of 100% methanol. Discard the solution and air dry the protein pellet.
12. Resuspend protein pellets into 600 µL of digestion buffer (12 mM sodium deoxycholate (SDC)/12 mM sodium lauroyl sarcosinate (SLS) in 100 mM Tris-HCl, pH 8.5). Sonicate the tube until the suspension is homogenized.
13. Measure the protein concentration using a BCA kit and adjust the concentration to 4 µg/µL with the digestion buffer. Transfer 100 µL of the lysate (400 µg proteins) into a new tube.
14. Add 292 µL of 50 mM triethylammonium bicarbonate (TEAB) and 8 µL of Lys-C (2.5 Unit/mL) to the tube. Incubate the tube at 37 °C for 3 h.
15. Add 100 µL of 50 mM TEAB with 8 µg trypsin to the tube. Incubate the tube at 37 °C for 12 h.
16. Add 25 µL of 10% trifluoroacetic acid (TFA) to the tube. Vortex and centrifuge the tube for 20 min at 4 °C.
17. Transfer the supernatant into a conditioned desalting column for desalting. Dry the eluate using a vacuum centrifuge concentrator.
    NOTE: Activate the resin with 1 mL of methanol followed by 1 mL of 0.1% TFA in 80% acetonitrile (ACN). Wash out the organic solvent with at least 3 mL of 0.1% TFA in 5% ACN. Load acidified peptide samples prepared in step 1.16 into the column and then wash the column with at least 3 mL of 0.1% TFA in 5% ACN. More washes may be needed for samples with high salt concentrations. Elute the phosphopeptides with 1 mL of 0.1% TFA in 80% ACN.

2. Tandem Mass Tag (TMT) labeling

1. Resuspend the dried peptides into 100 µL of 200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.5.
2. Dissolve the TMT reagent of each channel (0.8 mg) in 40 µL of anhydrous ACN. Vortex the TMT reagent tube for 5 min and spin it down.
3. Transfer 40 µL of TMT to the sample tube and incubate for 1 h at room temperature.
   NOTE: In this experiment, the channel 126 to 128 were labeled with three biological replicates of plants without mannitol treatment, and the channel 129 to 131 were labeled with three biological replicates of plants with mannitol treatment.
4. Add 8 µL of 5% hydroxylamine and incubate for 15 min.
5. Mix 6 samples in a 5 mL tube and add 14 µL of 10% TFA.
6. Add 3,876 µL of 0.1% TFA to the tube. Vortex and spin down.
7. Transfer the solution to a conditioned desalting column for desalting. Dry the eluate using an evaporator.

3. Preparation of IMAC stage tip

1. Use a 16 G blunt-ended needle to penetrate a polypropylene frit disk.
   NOTE: Hold the cutter perpendicular to the surface of the disk and roll the cutter a couple of times to make sure that the disk is completely excised. Place the disk in a Petri dish for storage. Place the needle into a 200 µL pipette tip and push the frit into the tip using a plunger.
2. Press the frit gently into the tip using a plunger to cap the end of tip.
   1. Place the frit disk in tips with the same pressure, which provides better reproducibility. The reproducibility of stage tips production is important for constant back pressure, which affects the timing of the centrifuge steps and reproducibility between technical replicates.
   2. If the stage tips show high back pressure during conditioning step, discard the tips to avoid potential tip clogging when while loading samples. It could be that the disk might have been pressed too hard into position.
3. Invert Ni-NTA spin column and place on a 1.5 mL tube.
4. Use a plunger to press the frit of Ni-NTA beads gently and push the beads into the tube.
   NOTE: Ensure to push the frit gently to avoid the potential bead loss or adsorption on the spin column.

4. Preparation of Hp-RP stage tip

1. Prepare Hp-RP stage tip as described for IMAC stage tip using a C8 disk.
   NOTE: Do not apply large force to the disk because it may result in a densely packed frit and increase the back pressure of the stage tip. All centrifugation steps take place at 1,000 x g for 5 min in step 4.
2. Suspend 1 mg of C18 beads in 100 µL 100% methanol and pass the beads solution through the tip.
3. Add 20 µL of buffer 8 (80% ACN/20% 200 mM NH₄HCO₂, pH 10.0) to the stage tip and pass buffer 8 through the tip by centrifugation.
4. Add 20 µL of Buffer A (200 mM NH₄HCO₂) to the stage tip and pass buffer A through the tip by centrifugation.
   NOTE: Balance the centrifuge during use and ensure that the Hp-RP stage tip is not completely dried.

5. Preparation of spin adaptor

1. Use sharp end tweezers to puncture a hole at the center of the lid of a 1.5 mL tube.
2. Insert the IMAC stage tip or Hp-RP stage tip into the hole of the spin adaptor.
   NOTE: Ensure that the stage tip is not close to the bottom of spin adaptor.

6. Phosphopeptide enrichment using IMAC stage tip

1. Suspend the 10 mg Ni-NTA beads with 400 µL of loading buffer (6% acetic acid (AA), pH 3.0) and load all of the beads solution into per tip. Pass the solution through the tip by centrifugation.
   NOTE: All centrifugation steps take place at 200 x g for 3 min in step 6 besides step 6.8.
2. Load 100 µL of 50 mM ethylenediaminetetraacetic acid (EDTA) to strip the nickel ions from IMAC stage tip and pass the solution through the tip by centrifugation.
3. Load 100 µL of loading buffer to the stage tip and pass the solution through the tip by centrifugation.
4. Load 100 µL of 50 mM FeCl₃ in 6% AA to the stage tip and pass the solution through the tip by centrifugation. Fe³⁺ ions will chelate to IMAC stage tip.
5. Load 100 µL of loading buffer to condition the stage tip and pass the solution through the tip by centrifugation.
6. Load 100 µL of loading buffer with sample peptides prepared in step 2.7 to the stage tip and pass the solution through the tip by centrifugation.
   NOTE: Take a new tube as a spin adaptor to collect the flow-through of sample and store the flow-through in the freezer for future analysis.
7. Load 100 µL of washing buffer (4.5% AA and 25% ACN) to the stage tip and pass the solution through the tip by centrifugation.
8. Perform the second wash with loading buffer. Load 100 µL of loading buffer to the stage tip and pass the solution through the tip using 1000 × g centrifuge for 3 min.
   NOTE: Ensure the washing buffer is replaced by loading buffer in the stage tip to eliminate the loss of phosphopeptides during elution step. Equilibrate the IMAC stage tip prior to phosphopeptide elution to ensure the concentration of ACN is below 5%.
9. Trim the IMAC stage tip at the front with a scissors and place the trimmed IMAC stage tip inside the Hp-RP tip. Ensure that the two layers of stage tips do not touch the lid of the centrifuge.

7. Phosphopeptide fractionation using Hp-RP C18 stage tip

1. Add 100 µL of elution buffer (200 mM ammonium phosphate (NH₄H₂PO₄)) to the trimmed IMAC stage tip and pass the solution through the two layers of stage tips by centrifugation.
   NOTE: If the solution does not pass through the stage tip, increase the speed of spin down. All centrifugation steps take place at 1,000 x g for 5 min in step 7. All the Hp-RP buffers are listed in Table 1.
2. Discard the IMAC stage tip using a tweezers and add 20 µL of Buffer A to the Hp-RP stage tip and pass the buffer through the tip by centrifugation.
   NOTE: Ensure all the elution buffer pass through the IMAC stage tip before discarding it.
3. Add 20 µL of Buffer 1 to elute fraction 1 and collect the eluate in a new tube by centrifugation. Repeat the process with Buffers 2, 3, 4, 5, 6, 7, and 8 to collect each fraction in a new tube. Dry the final eluate of each fraction using a vacuum concentrator.
   NOTE: Prepare fresh fractionation buffers. Take 8 new tubes as the spin adaptor of each fraction. Collect the eluate of each fraction in a different spin adaptor. Phosphopeptides are not stable under basic conditions, so dry the eluates by a concentrator or acidified by 10% TFA immediately.

8. LC-MS/MS analysis and data analysis

1. Add 5 µL of 0.1% formic acid (FA) and analyze the sample by a mass spectrometer.
2. Run a 90 min gradient with 6-30% buffer B (80% ACN and 0.1% FA) for each fraction.
3. Fragment the top 10 labeled peptides using high collision energy dissociation (HCD). Complete a database search to identify phosphorylation sites.
4. Load the raw files into the MaxQuant software, name the experiments, and set fractions and PTM.
   NOTE: The tutorial of MaxQuant software can be found at https://www.maxquant.org/.
5. Select reporter ion MS2 in the type of LC-MS/MS run and 6plex TMT as isobaric labels. Enable filter by PIF function and set the min PIF as 0.75.
6. Select Acetyl (Protein N-term), Oxidation (M), and Phospho (STY) to the panel of variable modifications. Select Carbamidomethyl (C) as fixed modifications.
7. Set digestion mode as specific, select Trypsin/P as digestion enzyme, and two missed cleavage.
8. Set PSM false discovery rate (FDR) and protein FDR as 0.01. Set the minimum score for modified peptides as 40.
9. Add Arabidopsis thaliana database to the panel of fasta files and run database searching.
10. Load the txt file of Phospho (STY) sites to the Perseus software as the search is done.
    NOTE: The detailed tutorials of Perseus software can be found at https://www.maxquant.org/perseus/.
11. Filter out the reverse phosphorylation sites. Filter out the unlocalized phosphorylation sites using localization probability 75% as the cut-off.
12. Use the intensities of phosphorylation sites for statistical analysis.

Wyniki

To demonstrate the performance of this workflow, we exploited IMAC stage tip coupled with Hp-RP stage tip fractionation to measure the phosphoproteomic changes in wild-type and snrk2-dec mutant seedlings with or without mannitol treatment for 30 minutes. Each sample was performed in biological triplicates, and the experimental workflow is represented in Figure 1. The digested peptides (400 µg) of each sample were labeled with one TMT-6plex channel, pooled and desalted. The phos...

Dyskusje

The dynamic range and complexity of plant proteome and phosphoproteome are still a limiting factor to depth of phosphoproteomics analyses. Despite the capability of single run LC-MS/MS analysis to identify 10,000 phosphorylation sites^21,22, the coverage of the whole plant phosphoproteome is still limited. Therefore, a phosphoproteomic workflow that provides high sensitivity and superior separation efficiency is required in profiling the global view of plant signa...

Materiały

Name	Company	Catalog Number	Comments
1.5 mL tube	eppendorf	22431081	Protein LoBind, 1.5 mL, PCR clean, colorless, 100 tubes
200 µL pipet tip	Gilson	F1739311
2-chloroacetamide	Sigma-Aldrich	C0267
acetic acid	Sigma-Aldrich	5438080100
acetonitrile	Sigma-Aldrich	271004
ammonium hydroxide	Sigma-Aldrich	338818
ammonium phosphate monbasic	Sigma-Aldrich	216003
BCA Protein Assay Kit	Thermo Fisher Scientific	23227
blunt-ended needle	Hamilton	90516	Kel-F hub (KF), point style 3, gauge 16
C18-AQ beads	Dr. Maisch	ReproSil-Pur-C18-AQ 5 µm
C8 Empore disk	3 M	2214	47 mm
Centrifuge	eppendorf	22620444
chloroform	Sigma-Aldrich	CX1058
data analysis software		Perseus 1.6.2.1	https://maxquant.net/perseus/
ethylenediaminetetraacetic acid (EDTA)	Sigma-Aldrich
formic acid	Sigma-Aldrich	5330020050
Frits	Agilent	12131024	Frits for SPE Cartridges
Guanidine hydrochloride	Sigma-Aldrich	50933
H2O	Sigma-Aldrich	1153334000
HEPES	Sigma-Aldrich	H3375
Iron (III) chloride	Sigma-Aldrich	157740
LTQ-orbitrap	Thermo Fisher Scientific	Velos Pro
mass spectrometer	Thermo Fisher Scientific	LTQ-Orbitrap Velos Pro
methanol	Sigma-Aldrich	34860
nano LC	Thermo Fisher Scientific	Easy-nLC 1000
Ni-NTA spin column	Qiagen	31014
N-Lauroylsarcosine sodium salt	Sigma-Aldrich	L9150
plunger	Hamilton	1122-01	Plunger assembly N, RN, LT, LTN for model 1702 (25 μl)
search engine software		MaxQuant 1.5.4.1	https://www.maxquant.org
SEP-PAK Cartridge 50 mg	Waters	WAT054960
sodium deoxycholate	Sigma-Aldrich	D6750
SpeedVac	Thermo Fisher Scientific	SPD121P
TMT 6-plex	Thermo Fisher Scientific	90061
Triethylammonium bicarbonate buffer	Sigma-Aldrich	T7408
Trifluoroacetic acid	Sigma-Aldrich	91707
Tris(2-carboxyethyl)phosphine hydrochloride	Sigma-Aldrich	C4706
Trizma hydrochloride	Sigma-Aldrich	T3253