Article pubs.acs.org/jpr
Integrative Network Analysis of the Signaling Cascades in Seedling Leaves of Bread Wheat by Large-Scale Phosphoproteomic Profiling Dong-Wen Lv, Pei Ge, Ming Zhang, Zhi-Wei Cheng, Xiao-Hui Li, and Yue-Ming Yan* College of Life Science, Capital Normal University, 100048 Beijing, China S Supporting Information *
ABSTRACT: Here, we conducted the first large-scale leaf phosphoproteome analysis of two bread wheat cultivars by liquid chromatography-tandem mass spectrometry. Altogether, 1802 unambiguous phosphorylation sites representing 1175 phosphoproteins implicated in various molecular functions and cellular processes were identified by gene ontology enrichment analysis. Among the 1175 phosphoproteins, 141 contained 3−10 phosphorylation sites. The phosphorylation sites were located more frequently in the N- and C-terminal regions than in internal regions, and ∼70% were located outside the conserved regions. Conservation analysis showed that 90.5% of the phosphoproteins had phosphorylated orthologs in other plant species. Eighteen significantly enriched phosphorylation motifs, of which six were new wheat phosphorylation motifs, were identified. In particular, 52 phosphorylated transcription factors (TFs), 85 protein kinases (PKs), and 16 protein phosphatases (PPs) were classified and analyzed in depth. All the Tyr phosphorylation sites were in PKs such as mitogen-activated PKs (MAPKs) and SHAGGY-like kinases. A complicated cross-talk phosphorylation regulatory network based on PKs such as Snf1-related kinases (SnRKs), calcium-dependent PKs (CDPKs), and glycogen synthase kinase 3 (GSK3) and PPs including PP2C, PP2A, and BRI1 suppressor 1 (BSU1)-like protein (BSL) was constructed and was found to be potentially involved in rapid leaf growth. Our results provide a series of phosphoproteins and phosphorylation sites in addition to a potential network of phosphorylation signaling cascades in wheat seedling leaves. KEYWORDS: bread wheat, leaf, phosphoproteome, kinases and phosphatases, regulatory network
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In recent years, ∼1000 PKs have been identified in most model plants whose genomes were sequenced, that is, ∼2-fold of those found in human or animal genomes.28 In eukaryotes, phosphorylation mainly occurs at serine (Ser), threonine (Thr), and tyrosine (Tyr) residues. In particular, Tyr phosphorylation plays crucial roles in the development of animals and humans, but it occurs far less frequently than Ser and Thr phosphorylation. For example, in human cells, phosphorylated Tyr residues account for only 2% of the total phosphorylated sites.29,30 Tyr phosphorylation in humans is mainly performed by Tyr-specific PKs (TKs). Additionally, Tyr can also be phosphorylated by dual-specificity PKs (DSKs), which possess both Ser/Thr and Tyr phosphorylation activity. TKs are thought to be absent in plants, but the proportion of phosphorylated Tyrs in plants is similar to that in humans.17,21 Tyr phosphorylation in plants may be performed through mechanisms different from those in humans. Little information is available regarding which PKs are involved in the phosphorylation of their respective substrates in plants. Interactions of PKs with their substrates are always determined by the residues surrounding the phosphorylation sites, the pattern of which is termed the phosphorylation motif.31,32 Motif-X analysis of large phosphorylation site data
INTRODUCTION
Protein phosphorylation is one of the most critical posttranslational modifications involved in the development and regulation of diverse processes, including metabolism, transcription and translation, protein degradation, homeostasis, cellular signaling and communication, proliferation, differentiation, and cell survival in various organisms.1−3 To date, various techniques have been developed for specific enrichment of phosphopeptides, such as Fe3+-IMAC (immobilized metal affinity chromatography)4,5 and TiO2-MOAC (metal oxide affinity chromatography).6,7 The principles of IMAC and MOAC are identical and both techniques use a positively charged chromatography matrix that binds to negatively charged phosphopeptides. MOAC protocols use modified buffers containing saturated solutions of organic acids, such as glycolic acid or lactic acid, to decrease nonspecific binding of acidic peptides.8,9 Recently, large-scale phosphoproteomic analyses of plants such as Arabidopsis thaliana,10−17,19,20 Oryza sativa,17,18 Medicago truncatula,21,22 Glycine max,19,23 Brassica napus,19 Zea mays,24−26 and Brachypodium distachyon27 have been performed using phosphopeptide enrichment combined with high-accuracy mass spectrometry (MS) and related bioinformatics tools. However, limited knowledge is available for phosphorylation modifications in other plant species, particularly in wheat. © 2014 American Chemical Society
Received: December 2, 2013 Published: March 19, 2014 2381
dx.doi.org/10.1021/pr401184v | J. Proteome Res. 2014, 13, 2381−2395
Journal of Proteome Research
Article
were vortexed thoroughly for 30 s, and the phenol phase was separated by centrifugation at 14 000 × g at 4 °C for 15 min. The upper phenol phase was pipetted into fresh 10 mL tubes; four volumes of cold methanol and 100 mM ammonium acetate were added, and the mixture was stored at −20 °C for at least 30 min. After the mixture was centrifuged at 14 000 × g at 4 °C for 15 min, the supernatant was carefully discarded, and the precipitated proteins were washed twice with cold methanolic ammonium acetate and then twice with 80% icecold acetone. The pellet was vacuum dried and then dissolved in lysis buffer (7 M urea, 2 M thiourea, 4% w/v CHAPS, and 65 mM DTT) for 3 h at 4 °C. The protein mixtures were harvested by centrifugation at 14 000 × g at 4 °C for 15 min to remove insoluble materials. The concentration of the extracted protein mixtures was determined with the 2-D Quant Kit (Amersham Bioscience, Buckinghamshire, UK) using bovine serum albumin (BSA; 2 mg/mL) as the standard. All the protein solutions were adjusted to a consistent concentration by using lysis buffer. The final protein solution was stored at −80 °C for later use.
sets can detect significantly enriched phosphorylation motifs and predict the corresponding PKs.33 Many large-scale phosphoproteomic analyses have used Motif-X to identify specific phosphorylation motifs in different conditions in various species, indicating that Motif-X is a powerful tool for identifying significant and novel phosphorylation motifs.20,34−36 Bread wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD), one of the most important food crops worldwide, provides ∼20% of the calories consumed by humans.37 Hexaploid bread wheat originated from a natural hybrid between the cultivated tetraploid wheat T. turgidum (2n = 4x = 28, AABB) and wild diploid grass Aegilops tauschii (2n = 14, DD).38,39 The large size of the wheat genome (∼17 Gb) hinders further genomic and proteomic study. However, along with the rapid development of genomics, the wheat genome project has made great progress. In particular, the recent release of the whole-genome shotgun sequencing data for bread wheat and its progenitor A and D genomes40−42 will greatly accelerate wheat proteome and phosphoproteome studies because protein peptides and phosphopeptide identification in addition to phosphosite localization largely depend on the homology-based matching of mass spectra to databases. In this study, we used TiO2 microcolumns to purify the phosphopeptides and liquid chromatography-tandem MS (LC−MS/MS) to identify the phosphorylation sites. To our knowledge, this is the first study in which large-scale phosphoproteomic analysis was performed for the seedling leaves of two Chinese bread wheat cultivars, Hanxuan 10 (HX10) and Ninchun 47 (NC47). Our results provide an overview of in vivo phosphorylation events and reveal a potential PK- and PP-based phosphorylation regulatory network during the rapid growth stage of seedling leaves in bread wheat.
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Phosphopeptide Enrichment Using TiO2 Microcolumns
The same amount of extracted protein mixture of each sample was directly reduced with DTT, alkylated with iodoacetamide, and subsequently digested with endoproteinase Lys-C and trypsin as previously described.30 The enrichment procedure for the phosphopeptides was performed as reported by Wu et al.7 with modifications. TiO2 beads (GL Sciences, Tokyo, Japan) were incubated in 400 μL of loading buffer containing 65% acetonitrile (ACN)/2% trifluoroaceticacid (TFA) saturated with glutamic acid. Tryptic peptides (2 mg) were dissolved in 600 μL of loading buffer and incubated with an appropriate amount (tryptic peptide:TiO2 = 1:1, w/w) of TiO2 beads. After washing with 600 μL of wash buffer (65% ACN/ 0.1% TFA), the phosphopeptides were eluted twice with 300 μL of elution buffer (500 mM NH4OH/60% ACN), and the eluates were dried and reconstituted in 0.1% formic acid (FA)/ H2O for MS analysis.
MATERIALS AND METHODS
Plant Materials
Seeds of HX10 and NC47 bread wheat cultivars, which are recently released elite bread wheat cultivars in Northern China, were surface-sterilized in 5% sodium hypochlorite for 5 min and rinsed four times in sterile distilled water. After submersion in water for 12 h at room temperature, the seeds were transferred to a wet filter paper to germinate at room temperature (22−25 °C) for 24 h. For each of the two bread wheat cultivars, ∼300 uniformly germinated seeds were selected and grown in each of the three plastic pots containing Hoagland’s solution, which was changed every 2 d. Wheat seedlings were cultured with three biological replicates. At the three-leaf stage, all three leaves of the seedlings were collected and frozen at −80 °C prior to analysis.
Phosphopeptide Identification and Phosphorylation Site Localization Using the LC−MS/MS and MaxQuant Software
The enriched phosphopeptides were separated on a self-packed C18 reverse-phase column (I.D., 75 μm; length, 150 mm; Column Technology Inc., Fremont, CA), which directly connected the nanoelectrospray ion source to an LTQ-Orbitrap XL mass spectrometer (Thermo Fisher Scientific, San Jose, CA). The pump flow was split to achieve a flow rate of 1 μL/ min for sample loading and 300 nL/min for MS analysis. The mobile phases consisted of 0.1% FA (A) and 0.1% FA/80% ACN (B). A five-step linear gradient from 5% to 30% B in 105 min, 35% to 90% B in 16 min, 90% B in 4 min, 90% to 2% B for 0.5 min, and 2% B for 14.5 min was applied. The spray voltage was set to 2.0 kV, and the temperature of the heated capillary was 240 °C. Each MS scan was acquired at a resolution of 60 000 (at 400 m/z) with the lock-mass option enabled, followed by top 10 data-dependent MS/MS scans performed using collisioninduced dissociation (CID). The threshold for precursor ion selection was 500, and the mass window for precursor ion selection was 2.0 Da. The dynamic exclusion duration was 120 s, the repeat count was 1, and the repeat duration was 30 s. The analyzer for the MS scans and MS/MS scans was Orbitrap and LTQ, respectively (37% relative collision energy). Three
Protein Extraction
Leaf total proteins were extracted according to the method reported by Wang et al.43 with minor modifications. Approximately 400 mg of fresh leaves of each sample was ground into fine powder in liquid nitrogen. The ground powder was suspended in 4 mL of SDS buffer (30% sucrose, 2% sodium dodecyl sulfate [SDS], 100 mM Tris-HCl, pH 8.0, 50 mM sodium-ethylenediaminetetraacetic acid [EDTA-Na2], and 20 mM dithiothreitol [DTT]) and 4 mL of phenol (Tris-buffered, pH 8.0) in a 10 mL tube, followed by the addition of 1 mM phenylmethanesulfonyl fluoride (PMSF) and PhosSTOP phosphatase-inhibitor cocktail (Roche, Basel, Switzerland) to inhibit the activity of proteases and phosphatases. The mixtures 2382
dx.doi.org/10.1021/pr401184v | J. Proteome Res. 2014, 13, 2381−2395
Journal of Proteome Research
Article
Figure 1. Strategy for a large-scale phosphoproteomics study on the leaves of the two bread wheat cultivars HX10 and NC47.
depending on their PTM localization probabilities,21,30 namely, class I (localization probability, P ≥ 0.75), class II (0.75 > P ≥ 0.5), and class III (P < 0.5). An FDR of 1% was used for phosphorylation site identification.
biological replicates were performed independently from sample collection to phosphopeptide identification. The raw files were processed with MaxQuant (version 1.2.2.5)44 and searched against the wheat protein database (77 037 entries) concatenated with a decoy consisting of reversed sequences. The following parameters were used for database searches: cysteine carbamidomethylation was selected as a fixed modification; methionine oxidation, protein N-terminal acetylation, and phosphorylation on Ser, Thr, and Tyr were selected as variable modifications. Up to two missing cleavage points were allowed. The precursor ion mass tolerances were 7 ppm, and the fragment ion mass tolerance for the MS/MS spectra was 0.5 Da. The false discovery rate (FDR) was set to
