This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant XDB27040106.

1. Sample preparation
<ol>
	<li>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.</li>
	<li>Grind frozen seedlings into a fine powder using a mortar and pestle filled with liquid nitrogen.</li>
	<li>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.</li>
	<li>Transfer the plant lysate to a 1.5 mL tube and heat at 95 &#176;C for 5 min.</li>
	<li>Place the tube on ice for 10 min. Sonicate the tube on ice for 10 s, pause for 10 s, and repeat thrice.</li>
	<li>Centrifuge the tube for 20 min and aliquot 150 &#181;L of plant lysate into a 1.5 mL tube.</li>
	<li>Add 600 &#181;L of 100% methanol into the tube. Vortex and spin down the tube.</li>
	<li>Add 150 &#181;L of 100% chloroform into the tube. Vortex and spin down the tube.</li>
	<li>Add 450 &#181;L of ddH2O into the tube. Vortex and centrifuge the tube for 3 min.</li>
	<li>Discard the upper aqueous layer. Add 600 &#181;L of 100% methanol into the tube. Centrifuge the tube for 3 min and then discard the solution.</li>
	<li>Wash protein pellets with 600 &#181;L of 100% methanol. Discard the solution and air dry the protein pellet.</li>
	<li>Resuspend protein pellets into 600 &#181;L 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.</li>
	<li>Measure the protein concentration using a BCA kit and adjust the concentration to 4 &#181;g/&#181;L with the digestion buffer. Transfer 100 &#181;L of the lysate (400 &#181;g proteins) into a new tube.</li>
	<li>Add 292 &#181;L of 50 mM triethylammonium bicarbonate (TEAB) and 8 &#181;L of Lys-C (2.5 Unit/mL) to the tube. Incubate the tube at 37 &#176;C for 3 h.</li>
	<li>Add 100 &#181;L of 50 mM TEAB with 8 &#181;g trypsin to the tube. Incubate the tube at 37 &#176;C for 12 h.</li>
	<li>Add 25 &#181;L of 10% trifluoroacetic acid (TFA) to the tube. Vortex and centrifuge the tube for 20 min at 4 &#176;C.</li>
	<li>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.</li>
</ol>
2. Tandem Mass Tag (TMT) labeling
<ol>
	<li>Resuspend the dried peptides into 100 &#181;L of 200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.5.</li>
	<li>Dissolve the TMT reagent of each channel (0.8 mg) in 40 &#181;L of anhydrous ACN. Vortex the TMT reagent tube for 5 min and spin it down.</li>
	<li>Transfer 40 &#181;L 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.</li>
	<li>Add 8 &#181;L of 5% hydroxylamine and incubate for 15 min.</li>
	<li>Mix 6 samples in a 5 mL tube and add 14 &#181;L of 10% TFA.</li>
	<li>Add 3,876 &#181;L of 0.1% TFA to the tube. Vortex and spin down.</li>
	<li>Transfer the solution to a conditioned desalting column for desalting. Dry the eluate using an evaporator.</li>
</ol>
3. Preparation of IMAC stage tip
<ol>
	<li>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 &#181;L pipette tip and push the frit into the tip using a plunger.</li>
	<li>Press the frit gently into the tip using a plunger to cap the end of tip.
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Invert Ni-NTA spin column and place on a 1.5 mL tube.</li>
	<li>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.</li>
</ol>
4. Preparation of Hp-RP stage tip
<ol>
	<li>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.</li>
	<li>Suspend 1 mg of C18 beads in 100 &#181;L 100% methanol and pass the beads solution through the tip.</li>
	<li>Add 20 &#181;L 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.</li>
	<li>Add 20 &#181;L 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.</li>
</ol>
5. Preparation of spin adaptor
<ol>
	<li>Use sharp end tweezers to puncture a hole at the center of the lid of a 1.5 mL tube.</li>
	<li>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.</li>
</ol>
6. Phosphopeptide enrichment using IMAC stage tip
<ol>
	<li>Suspend the 10 mg Ni-NTA beads with 400 &#181;L 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.</li>
	<li>Load 100 &#181;L of 50 mM ethylenediaminetetraacetic acid (EDTA) to strip the nickel ions from IMAC stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Load 100 &#181;L of loading buffer to the stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Load 100 &#181;L 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.</li>
	<li>Load 100 &#181;L of loading buffer to condition the stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Load 100 &#181;L 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.</li>
	<li>Load 100 &#181;L of washing buffer (4.5% AA and 25% ACN) to the stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Perform the second wash with loading buffer. Load 100 &#181;L of loading buffer to the stage tip and pass the solution through the tip using 1000 &#215; 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%.</li>
	<li>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.</li>
</ol>
7. Phosphopeptide fractionation using Hp-RP C18 stage tip
<ol>
	<li>Add 100 &#181;L 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.</li>
	<li>Discard the IMAC stage tip using a tweezers and add 20 &#181;L 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.</li>
	<li>Add 20 &#181;L 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.</li>
</ol>
8. LC-MS/MS analysis and data analysis
<ol>
	<li>Add 5 &#181;L of 0.1% formic acid (FA) and analyze the sample by a mass spectrometer.</li>
	<li>Run a 90 min gradient with 6-30% buffer B (80% ACN and 0.1% FA) for each fraction.</li>
	<li>Fragment the top 10 labeled peptides using high collision energy dissociation (HCD). Complete a database search to identify phosphorylation sites.</li>
	<li>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/.</li>
	<li>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.</li>
	<li>Select Acetyl (Protein N-term), Oxidation (M), and Phospho (STY) to the panel of variable modifications. Select Carbamidomethyl (C) as fixed modifications.</li>
	<li>Set digestion mode as specific, select Trypsin/P as digestion enzyme, and two missed cleavage.</li>
	<li>Set PSM false discovery rate (FDR) and protein FDR as 0.01. Set the minimum score for modified peptides as 40.</li>
	<li>Add Arabidopsis thaliana database to the panel of fasta files and run database searching.</li>
	<li>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/.</li>
	<li>Filter out the reverse phosphorylation sites. Filter out the unlocalized phosphorylation sites using localization probability 75% as the cut-off.</li>
	<li>Use the intensities of phosphorylation sites for statistical analysis.</li>
</ol>

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The authors declare no conflict of interest.

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 sites21,22, 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...

High salinity, low temperature, and drought cause osmotic stresses, which is a major environmental factor that affects plant productivity1,2. Protein phosphorylation is one of the most significant post-translational modifications mediating signal perception and transduction in plant response to osmotic stress3,4,5. 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 stress7,8. 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 analysis11,12,13,14,15,16. 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.

<table>
	<tbody>
		<tr>
			<td>1.5 mL tube</td>
			<td>eppendorf</td>
			<td>22431081</td>
			<td>Protein LoBind, 1.5 mL, PCR clean, colorless, 100 tubes</td>
		</tr>
		<tr>
			<td>200 &#181;L pipet tip</td>
			<td>Gilson</td>
			<td>F1739311</td>
			<td></td>
		</tr>
		<tr>
			<td>2-chloroacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>C0267</td>
			<td></td>
		</tr>
		<tr>
			<td>acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>5438080100</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>271004</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>338818</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium phosphate monbasic</td>
			<td>Sigma-Aldrich</td>
			<td>216003</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr>
			<td>blunt-ended needle</td>
			<td>Hamilton</td>
			<td>90516</td>
			<td>Kel-F hub (KF), point style 3, gauge 16</td>
		</tr>
		<tr>
			<td>C18-AQ beads</td>
			<td>Dr. Maisch</td>
			<td>ReproSil-Pur-C18-AQ 5 &#181;m</td>
			<td></td>
		</tr>
		<tr>
			<td>C8 Empore disk</td>
			<td>3 M</td>
			<td>2214</td>
			<td>47 mm</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>eppendorf</td>
			<td>22620444</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>CX1058</td>
			<td></td>
		</tr>
		<tr>
			<td>data analysis software</td>
			<td></td>
			<td>Perseus 1.6.2.1</td>
			<td>https://maxquant.net/perseus/</td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Sigma-Aldrich</td>
			<td>5330020050</td>
			<td></td>
		</tr>
		<tr>
			<td>Frits</td>
			<td>Agilent</td>
			<td>12131024</td>
			<td>Frits for SPE Cartridges</td>
		</tr>
		<tr>
			<td>Guanidine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>50933</td>
			<td></td>
		</tr>
		<tr>
			<td>H2O</td>
			<td>Sigma-Aldrich</td>
			<td>1153334000</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (III) chloride</td>
			<td>Sigma-Aldrich</td>
			<td>157740</td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ-orbitrap</td>
			<td>Thermo Fisher Scientific</td>
			<td>Velos Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>LTQ-Orbitrap Velos Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>nano LC</td>
			<td>Thermo Fisher Scientific</td>
			<td>Easy-nLC 1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni-NTA spin column</td>
			<td>Qiagen</td>
			<td>31014</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>plunger</td>
			<td>Hamilton</td>
			<td>1122-01</td>
			<td>Plunger assembly N, RN, LT, LTN for model 1702 (25 &#956;l)</td>
		</tr>
		<tr>
			<td>search engine software</td>
			<td></td>
			<td>MaxQuant 1.5.4.1</td>
			<td>https://www.maxquant.org</td>
		</tr>
		<tr>
			<td>SEP-PAK Cartridge 50 mg</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D6750</td>
			<td></td>
		</tr>
		<tr>
			<td>SpeedVac</td>
			<td>Thermo Fisher Scientific</td>
			<td>SPD121P</td>
			<td></td>
		</tr>
		<tr>
			<td>TMT 6-plex</td>
			<td>Thermo Fisher Scientific</td>
			<td>90061</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethylammonium bicarbonate buffer</td>
			<td>Sigma-Aldrich</td>
			<td>T7408</td>
			<td></td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>91707</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
			<td></td>
		</tr>
	</tbody>
</table>

phosphoproteomic strategy for profiling osmotic stress signaling in arabidopsis

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 (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 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.

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 &#181;g) of each sample were labeled with one TMT-6plex channel, pooled and desalted. The phos...

Watch this Scientific Journal Video about Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis at JoVE.com

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

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.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

phosphoproteomic-strategy-for-profiling-osmotic-stress-signaling

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Biochemistry

5.1K Views.  Carnegie Institute for Science. 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.

Video: Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

Sheathless Capillary Electrophoresis&amp;#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture (CARIC) Strategy

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used strategy for RBP capture exploits the polyadenylation [poly(A)] of target RNAs, which mostly occurs on eukaryotic mature mRNAs, leaving most binding proteins of non-poly(A) RNAs unidentified. Here we describe the detailed procedures of a recently reported method termed click chemistry-assisted RNA-interactome capture (CARIC), which enables the transcriptome-wide capture of both poly(A) and non-poly(A) RBPs by combining the metabolic labeling of RNAs, in vivo UV cross-linking, and bioorthogonal tagging.

Capture and Identification of RNA-binding Proteins by Using Click Chemistry-assisted RNA-interactome Capture CARIC Strategy

A detailed protocol for applying the click chemistry-assisted RNA-interactome capture (CARIC) strategy to identify proteins binding to both coding and noncoding RNAs is presented.

A comprehensive identification of RNA-binding proteins (RBPs) is key to understanding the posttranscriptional regulatory network in cells. A widely used ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

Fabrication of Streptavidin Affinity Grids for Cryo-EM Structural Studies

Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, including sample denaturation and preferential orientations that can occur due to the air-water interface. Streptavidin affinity grids, however, are currently utilized by few cryo-EM labs because they are not commercially available and require a careful fabrication process. Two-dimensional streptavidin crystals are grown onto a biotinylated lipid monolayer that is applied directly to standard holey-carbon cryo-EM grids. The high-affinity interaction between streptavidin and biotin allows for the subsequent binding of biotinylated samples that are protected from the air-water interface during cryo-EM sample preparation. Additionally, these grids provide a strategy for concentrating samples available in limited quantities and purifying protein complexes of interest directly on the grids. Here, a step-by-step, optimized protocol is provided for the robust fabrication of streptavidin affinity grids for use in cryo-EM and negative-stain experiments. Additionally, a trouble-shooting guide is included for commonly experienced challenges to make the use of streptavidin affinity grids more accessible to the larger cryo-EM community.

Author Spotlight: Enhancing Cryo-EM Sample Preparation with Streptavidin-Biotin Approach

A step-by-step protocol for fabricating streptavidin affinity grids is provided for use in structural studies of challenging macromolecular samples by cryo-electron microscopy.

Streptavidin affinity grids provide strategies to overcome many commonly encountered cryo-electron microscopy (cryo-EM) sample preparation challenges, ...