Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Summary

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.

Abstract

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.

Introduction

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 stre....

Protocol

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-carboxyethy....

Results

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 phosphope.......

Discussion

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.......

Materials

List of materials used in this article

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