Universal Plant Phosphoproteomics Workflow and Its Application to Tomato Signaling in Response to Cold Stress*

Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species in response to a cold environment. The activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms. Phosphorylation-mediated signaling transduction plays a crucial role in the regulation of plant defense mechanisms against environmental stresses. To address the high complexity and dynamic range of plant proteomes and phosphoproteomes, we present a universal sample preparation procedure that facilitates plant phosphoproteomic profiling. This advanced workflow significantly improves phosphopeptide identifications, enabling deep insight into plant phosphoproteomes. We then applied the workflow to study the phosphorylation events involved in tomato cold tolerance mechanisms. Phosphoproteomic changes of two tomato species (N135 Green Gage and Atacames) with distinct cold tolerance phenotypes were profiled under cold stress. In total, we identified more than 30,000 unique phosphopeptides from tomato leaves, representing about 5500 phosphoproteins, thereby creating the largest tomato phosphoproteomic resource to date. The data, along with the validation through in vitro kinase reactions, allowed us to identify kinases involved in cold tolerant signaling and discover distinctive kinase-substrate events in two tomato species in response to a cold environment. The activation of SnRK2s and their direct substrates may assist N135 Green Gage tomatoes in surviving long-term cold stress. Taken together, the streamlined approach and the resulting deep phosphoproteomic analyses revealed a global view of tomato cold-induced signaling mechanisms. Protein phosphorylation is crucial for plant cells in perceiving and responding to environmental stimuli through transduction of signals from receptor kinases to targets (1Zhu J.K. Abiotic Stress Signaling and Responses in Plants.Cell. 2016; 167: 313-324Abstract Full Text Full Text PDF PubMed Scopus (2353) Google Scholar, 2Schulze W.X. Proteomics approaches to understand protein phosphorylation in pathway modulation.Curr. Opin. Plant Biol. 2010; 13: 280-287Crossref PubMed Scopus (64) Google Scholar). Compared with those of humans, plant kinases are double in number and diversity, highlighting the importance of the plant kinome and phosphoproteome in regulating responses to both abiotic and biotic stresses (3Silva-Sanchez C. Li H. Chen S. Recent advances and challenges in plant phosphoproteomics.Proteomics. 2015; 15: 1127-1141Crossref PubMed Scopus (84) Google Scholar, 4Singh D.K. Calvino M. Brauer E.K. Fernandez-Pozo N. Strickler S. Yalamanchili R. Suzuki H. Aoki K. Shibata D. 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Lee B.H. Hong X. Agarwal M. Zhu J.K. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1212) Google Scholar). Under cold stress, ice1 mutant plants had reduced expression of CBF genes and displayed the cold sensitive phenotype (9Chinnusamy V. Ohta M. Kanrar S. Lee B.H. Hong X. Agarwal M. Zhu J.K. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis.Genes Dev. 2003; 17: 1043-1054Crossref PubMed Scopus (1212) Google Scholar). Besides the important role of transcriptional factors in cold acclimation, many plant kinases are activated and positively regulate plant freezing tolerance at the post-translational level. One of the canonical events is the activation of MEKK1-MKK2-MAPK4/6 cascades in Arabidopsis under short periods of cold treatment, which has been linked to enhanced freezing tolerance (10Teige M. Scheikl E. Eulgem T. Doczi R. Ichimura K. Shinozaki K. 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Another example is the serine/threonine kinase OST1, one of the core components of the Abscisic acid (ABA) 1The abbreviations used are:ABAabscisic acidCAA2-chloroacetamideCKL2casein kinase 1 like protein 2FASPfilter aided sample preparationGdnHClguanidine hydrochlorideMAPKmitogen activated protein kinaseMWCOmolecular weight cut-off filterPolyMACpolymer-based metal-ion affinity capturePTSphase-transfer surfactantSDCsodium deoxycholatesiKALIPstable isotope labeling kinase assay linked phosphoproteomicsSLSsodium lauroyl sarcosinateSnRK2sucrose non-fermenting 1-related protein kinase 2TCEPTris(2-carboxyethyl)phosphine hydrochlorideTSAPtemperature-sensitive alkaline phosphatase. 1The abbreviations used are:ABAabscisic acidCAA2-chloroacetamideCKL2casein kinase 1 like protein 2FASPfilter aided sample preparationGdnHClguanidine hydrochlorideMAPKmitogen activated protein kinaseMWCOmolecular weight cut-off filterPolyMACpolymer-based metal-ion affinity capturePTSphase-transfer surfactantSDCsodium deoxycholatesiKALIPstable isotope labeling kinase assay linked phosphoproteomicsSLSsodium lauroyl sarcosinateSnRK2sucrose non-fermenting 1-related protein kinase 2TCEPTris(2-carboxyethyl)phosphine hydrochlorideTSAPtemperature-sensitive alkaline phosphatase. pathway, which modulates ICE1 protein turnover through phosphorylation at Ser278 (13Ding Y. Li H. Zhang X. Xie Q. Gong Z. Yang S. OST1 kinase modulates freezing tolerance by enhancing ICE1 stability in Arabidopsis.Dev. Cell. 2015; 32: 278-289Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar). Phosphorylation of this site prevents the degradation of ICE1 protein under cold stress, which promotes cold tolerance in Arabidopsis. These examples suggest that the plant phosphoproteome and kinome are involved in the regulation of molecular events that trigger cold acclimation. abscisic acid 2-chloroacetamide casein kinase 1 like protein 2 filter aided sample preparation guanidine hydrochloride mitogen activated protein kinase molecular weight cut-off filter polymer-based metal-ion affinity capture phase-transfer surfactant sodium deoxycholate stable isotope labeling kinase assay linked phosphoproteomics sodium lauroyl sarcosinate sucrose non-fermenting 1-related protein kinase 2 Tris(2-carboxyethyl)phosphine hydrochloride temperature-sensitive alkaline phosphatase. abscisic acid 2-chloroacetamide casein kinase 1 like protein 2 filter aided sample preparation guanidine hydrochloride mitogen activated protein kinase molecular weight cut-off filter polymer-based metal-ion affinity capture phase-transfer surfactant sodium deoxycholate stable isotope labeling kinase assay linked phosphoproteomics sodium lauroyl sarcosinate sucrose non-fermenting 1-related protein kinase 2 Tris(2-carboxyethyl)phosphine hydrochloride temperature-sensitive alkaline phosphatase. As the tomato is one of the most important horticultural crops in the world, we utilized our proteomic approach to investigate the underlying mechanisms of their cold tolerance. Considering the limited knowledge of the molecular mechanisms that regulate tomato cold tolerance, different tomato subtypes with distinct cold tolerances are suitable as a model system to study these mechanisms. Previous reports have systematically compared the cold tolerances of tomatoes in the view of the transcriptome (14Chen H. Chen X. Chen D. Li J. Zhang Y. Wang A. A comparison of the low temperature transcriptomes of two tomato genotypes that differ in freezing tolerance: Solanum lycopersicum and Solanum habrochaites.BMC Plant Biol. 2015; 15: 132Crossref PubMed Scopus (67) Google Scholar, 15Patade V.Y. Khatri D. Kumari M. Grover A. Mohan Gupta S. Ahmed Z. Cold tolerance in Osmotin transgenic tomato (Solanum lycopersicum L.) is associated with modulation in transcript abundance of stress responsive genes.Springerplus. 2013; 2: 117Crossref PubMed Scopus (37) Google Scholar). Distinct gene expression patterns were observed from different cold tolerant tomato variants, indicating the complexity of cold-induced molecular mechanisms in tomatoes. However, there has been no large-scale study to characterize the roles of the tomato kinome and phosphoproteome in the regulation of cold tolerance. We recently observed that a novel cultivated tomato, N135 Green Gage (cultivar, Solanum lycopersicum), is tolerant of prolonged cold exposure (4 °C), whereas a wild tomato, Atacames (PIM, Solanum. pimpinellifolium), displays a cold-sensitive phenotype under cold conditions. Thus, these tomatoes are ideal materials to study the important cold tolerance signaling pathways in tomato. Mass spectrometry (MS) has emerged as a powerful technology for identifying thousands of phosphorylation sites in a single shot from mammalian cells and extracellular vesicles (16Imamura H. Sugiyama N. Wakabayashi M. Ishihama Y. Large-scale identification of phosphorylation sites for profiling protein kinase selectivity.J. Proteome Res. 2014; 13: 3410-3419Crossref PubMed Scopus (38) Google Scholar, 17Tsai C.F. Hsu C.C. Hung J.N. Wang Y.T. Choong W.K. Zeng M.Y. Lin P.Y. Hong R.W. Sung T.Y. Chen Y.J. Sequential phosphoproteomic enrichment through complementary metal-directed immobilized metal ion affinity chromatography.Anal. Chem. 2014; 86: 685-693Crossref PubMed Scopus (83) Google Scholar, 18Humphrey S.J. Azimifar S.B. Mann M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.Nat. Biotechnol. 2015; 33: 990-995Crossref PubMed Scopus (310) Google Scholar, 19Chen I.H. Xue L. Hsu C.C. Paez J.S. Pan L. Andaluz H. Wendt M.K. Iliuk A.B. Zhu J.K. Tao W.A. Phosphoproteins in extracellular vesicles as candidate markers for breast cancer.Proc. Natl. Acad. Sci. U.S.A. 2017; 114: 3175-3180Crossref PubMed Scopus (245) Google Scholar). However, significant analytical difficulty is encountered in plant phosphoproteomics because of the high dynamic range of the plant proteome, the rigidity of plant cell walls, and the interference from chlorophyll and secondary metabolites (20Isaacson T. Damasceno C.M. Saravanan R.S. He Y. Catala C. Saladie M. Rose J.K. Sample extraction techniques for enhanced proteomic analysis of plant tissues.Nat. Protoc. 2006; 1: 769-774Crossref PubMed Scopus (359) Google Scholar, 21Wang W. Tai F. Chen S. Optimizing protein extraction from plant tissues for enhanced proteomics analysis.J. Sep. Sci. 2008; 31: 2032-2039Crossref PubMed Scopus (130) Google Scholar, 22Kim Y.J. Lee H.M. Wang Y. Wu J. Kim S.G. Kang K.Y. Park K.H. Kim Y.C. Choi I.S. Agrawal G.K. Rakwal R. Kim S.T. Depletion of abundant plant RuBisCO protein using the protamine sulfate precipitation method.Proteomics. 2013; 13: 2176-2179Crossref PubMed Scopus (54) Google Scholar). These challenges hamper the sensitivity and efficiency of detecting low abundance phosphorylation events in plants through MS. In addition, resources concerning the tomato phosphoproteome are still limited by the lack of a full genomic sequence of the tomato. Stulemeijer et al. only reported 50 phosphoproteins from tomato seedlings using TiO2 enrichment and LC-MS/MS analysis, which was partially attributed to poor spectrum and protein sequence matching (23Stulemeijer I.J. Joosten M.H. Jensen O.N. Quantitative phosphoproteomics of tomato mounting a hypersensitive response reveals a swift suppression of photosynthetic activity and a differential role for hsp90 isoforms.J. Proteome Res. 2009; 8: 1168-1182Crossref PubMed Scopus (41) Google Scholar). To address the limits of global plant phosphoproteomics, we have carefully evaluated the performance of several techniques that have been previously employed in each proteomic sample preparation step. We introduce here a universal sample preparation protocol, which significantly increased the coverage and depth of the plant phosphoproteome. This protocol was then applied to study the phosphoproteomic perturbation of two tomato varieties under prolonged cold treatment. This in-depth phosphoproteomic resource reveals the phosphorylation sites implicated in kinase activation and cold-responsive gene expression. Upon coupling this data with in vitro kinase screening, we discovered a connection between SnRK2s activation and cold tolerance through phosphorylation of their downstream kinases, which sheds light upon which tomato phosphoproteins are critical for conferring cold tolerance. We designed and compared six sample preparation workflows detailed in Table I. Three technical replicates were performed for each protocol, and one LC-MS/MS injection was performed for each replicate resulting in a total of 18 analyses. The Tris-HCl protocol was used as a standard control to compare the number of identified phosphopeptides with the other strategies. The number of unique phosphopeptides identified from each replicate of the strategies was calculated using Microsoft Excel after removal of the redundant phosphorylated sequences in the MaxQuant output evidence file.Table IComparison of the sample preparation protocols for tomato phosphoproteomicsProtocolAmountLysis bufferDepletion methodDigestion bufferLysis efficiency200 μg100 mm Tris-HClN/A100 mm Tris-HCl200 μg12 mm SDC-SLSN/A2.4 mm SDC-SLS200 μg6 m Gdn-HClN/A600 mm Gdn-HClInterferences depletion200 μg6 m Gdn-HCl10K DaaMolecular weight cut-off filter.50 mm ABC200 μg6 m Gdn-HClM.-C. precipitationbMethanol-chloroform precipitation.1 m UreaDigestion buffer200 μg6 m Gdn-HClM.-C. precipitation1 m Urea200 μg6 m Gdn-HClM.-C. precipitation2.4 mm SDC-SLSa Molecular weight cut-off filter.b Methanol-chloroform precipitation. Open table in a new tab To access the tomato phosphoproteome and to quantify the phosphoproteomic changes in terms of long-term cold stress (4 °C, 5 days), three biological replicates were collected from the 30-day-old leaves of N135 Green Gage and Atacames tomatoes treated maintained at either room temperature or 4 °C (cold stress) for 5 days, resulting in a total of 12 samples. Stable isotope dimethyl labeling was performed to quantify the phosphorylation perturbation upon cold stress. The leaves from plants treated at room temperature were set as controls and the corresponding peptides were labeled using light formaldehyde (CH2O), whereas the samples exposed to cold stress were set as treatments and labeled using heavy formaldehyde (C13D2O). The same amount of a control and a treatment replicate were pooled together, and the labeled phosphopeptides were purified and subsequently separated into six fractions. Each fraction was analyzed by one LC-MS/MS injection, resulting in a total of 36 raw files. The number of identified phosphopeptides was calculated as described above. The statistical analysis is described in the Data Analysis subsection. Tomatoes were grown in a greenhouse at Purdue University. Tomato seeds were first germinated in soil, and the seedlings with identical growth were transferred to separate pots. Four-week-old tomato plants (about 30 cm height) were subjected to mock or cold treatment (4 °C) for 5 days. Equal amounts of leaf tissue from cold tolerant and sensitive tomato varieties were collected for further electrolytic leakage and phosphoproteomic analyses. The electrolyte leakage (EL) was measured as follows: at least three fully expanded leaves from each tomato variety were detached and immersed in 50 ml tubes with 25 ml distilled water. After gentle shaking overnight, the initial electrolyte conductivity (E1) was measured with a conductivity meter. Next, the samples were autoclaved. After cooling to room temperature, the conductivity (E2) was measured again. The relative electrolyte leakage was calculated as: E1/E2 × 100. RNA was extracted from 1-month-old tomato plants grown in soil under mock treatment or 4 °C for the indicated time. 1 μg RNA was used for reverse transcription using M-MLV reverse transcriptase (Promega). Real-time PCR was performed using iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) on a CFX96 real-time PCR detection system (Bio-Rad). Tomato Actin 7 (ACT7) was used as the internal reference for all reactions. Ground tomato leaves were lysed in (1) Tris-HCl protocol: 100 mm Tris-HCl (pH 7.5), 250 mm NaCl, and 5 mm EDTA (24Wang P.C. Xue L. Batelli G. Lee S. Hou Y.J. Van Oosten M.J. Zhang H.M. Tao W.A. Zhu J.K. Quantitative phosphoproteomics identifies SnRK2 protein kinase substrates and reveals the effectors of abscisic acid action.Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 11205-11210Crossref PubMed Scopus (315) Google Scholar), (2) SDC-SLS protocol: 100 mm Tris-HCl (pH 8.5), 12 mm SDC, and 12 mm SLS (25Masuda T. Sugiyama N. Tomita M. Ishihama Y. Microscale phosphoproteome analysis of 10,000 cells from human cancer cell lines.Anal. Chem. 2011; 83: 7698-7703Crossref PubMed Scopus (61) Google Scholar), or (3) GdnHCl protocol: 100 mm Tris-HCl (pH 8.5) and 6 m guanidine chloride (GdnHCl) (18Humphrey S.J. Azimifar S.B. Mann M. High-throughput phosphoproteomics reveals in vivo insulin signaling dynamics.Nat. Biotechnol. 2015; 33: 990-995Crossref PubMed Scopus (310) Google Scholar). All three lysis buffers were supplemented with EDTA-free protease and phosphatase inhibitor cocktails (Sigma-Aldrich, St. Louis, MO). Protein amount was quantified by BCA assay (Thermo Fisher Scientific, Waltham, MA). Proteins were reduced and alkylated with 10 mm TCEP and 40 mm CAA at 95 °C for 5 min. A 200 μg aliquot of lysate was loaded into a Microcon 10k Da (MilliporeSigma, Burlington, MA) filter tube using the FASP protocol (26Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Methods. 2009; 6: 359-362Crossref PubMed Scopus (5097) Google Scholar). The GdnHCl buffer was replaced by 100 μl of 8 m urea buffer; The urea buffer was replaced by 100 μl 50 mm ammonium bicarbonate (ABC). Lys-C was added onto the filter at a 1:100 (w/w) ratio for 3 h at 37 °C, and trypsin was subsequently added to a final 1:100 (w/w) ratio overnight. The digests were collected with two washes of 100 μl 50 mm ABC buffer, and then the peptides were acidified with 10% trifluoroacetic acid (TFA) to pH ∼3 and desalted using a 50 mg Sep-Pak C18 column. The protein precipitation was performed as previously described with some modifications (27Wessel D. Flugge U.I. A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids.Anal. Biochem. 1984; 138: 141-143Crossref PubMed Scopus (3167) Google Scholar). First, 200 μg of lysate was added to four volumes of methanol, followed by an equal volume of chloroform with mixing. Three volumes of ddH2O were added to the tube with mixing. The solution was centrifuged at 16,000 × g for 3 min. The upper aqueous layer was removed, the protein pellet was washed with four volumes of methanol, and the tube was centrifuged again. The supernatant was discarded, and the precipitated protein pellet was air dried. Protein extracts (200 μg) were 5-fold diluted for the SDC-SLS protocol (28Masuda T. Tomita M. Ishihama Y. Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis.J. Proteome Res. 2008; 7: 731-740Crossref PubMed Scopus (417) Google Scholar), 8-fold diluted for the urea protocol, or 10-fold diluted for the GdnHCl protocol using 50 mm TEAB. Proteins were then digested with Lys-C (FUJIFILM Wako Chemicals, Richmond, VA) in a 1:100 (w/w) enzyme-to-protein ratio for 3 h at 37 °C, and trypsin (Sigma-Aldrich) was added to a final 1:100 (w/w) enzyme-to-protein ratio overnight. The SDC and SLS were separated from digested peptides by acidifying the solution using 10% TFA, adding 100%ethyl acetate (50% final volume), and removing the organic solution. The aqueous layer was dried in a SpeedVac, and digests were then suspended with 0.1% TFA and desalted using a 50 mg Sep-Pak C18 column (Waters, Milford, MA). Dimethyl-labeling was performed as previously described (29Boersema P.J. Raijmakers R. Lemeer S. Mohammed S. Heck A.J. Multiplex peptide stable isotope dimethyl labeling for quantitative proteomics.Nat. Protoc. 2009; 4: 484-494Crossref PubMed Scopus (1062) Google Scholar). The tryptic peptides were dissolved in 100 μl of 100 mm TEAB and mixed with 4 μl of 4%13CD2O or 12CH2O, and then 4 μl of freshly prepared 0.6 m sodium cyanoborohydride was immediately added. The mixture was agitated for 60 min at room temperature. The reaction was stopped by adding 16 μl of 1% ammonium hydroxide on ice and agitating the mixture for 1 min. The dimethyl labeled peptides were then mixed with 20 μl of 10% formic acid (FA), and the two labeled samples were combined and desalted using a Sep-Pak C18 column. Phosphopeptides were enriched using a PolyMAC-Ti kit (Tymora Analytical, West Lafayette, IN) (30Iliuk A.B. Martin V.A. Alicie B.M. Geahlen R.L. Tao W.A. In-depth analyses of kinase-dependent tyrosine phosphoproteomes based on metal ion-functionalized soluble nanopolymers.Mol. Cell. Proteomics. 2010; 9: 2162-2172Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Briefly, the digested peptides were resuspended with 20 μl of ultrapure water, 200 μl of Loading buffer (50 mm glycolic acid in 0.5% TFA and 95% ACN) was added, and the sample was vortexed. Next, 50 μl of the PolyMAC/Magnetic Capture beads were added to the sample and incubated for 20 min. The solvent was removed using a magnetic separator rack, and the beads were washed with 200 μl of Washing buffer 1 (1% TFA in 80% ACN) for 5 min. The beads were then incubated with 200 μl of Washing buffer 2 (80% ACN in ddH2O) for 5 min, and the solvent was removed as before. Finally, the beads were incubated twice with 100 μl of Elution buffer (400 mm NH4OH in 50% ACN) for 5 min each. The eluates were combined and completely dried in a SpeedVac. Basic pH reverse-phase fractionation was performed with some modifications as previously described (31Dimayacyac-Esleta B.R. Tsai C.F. Kitata R.B. Lin P.Y. Choong W.K. Lin T.D. Wang Y.T. Weng S.H. Yang P.C. Arco S.D. Sung T.Y. Chen Y.J. Rapid high-pH reverse phase StageTip for sensitive small-scale membrane proteomic profiling.Anal. Chem. 2015; 87: 12016-12023Crossref PubMed Scopus (36) Google Scholar). First, 2 mg of Magic C18-AQ beads (5 μm particle size) were suspended in 100 μl of methanol and loaded into a 200 μl StageTip with a 20 μm polypropylene frit. The C18 StageTips were activated with 100 μl of 40 mm NH4HCO2, pH 10, in 80% ACN and equilibrated with 100 μl of 200 mm NH4HCO2, pH 10. The isolated phosphopeptides were suspended with 200 mm NH4HCO2, and the C18 StageTips were washed with 100 μl of 200 mm NH4HCO2, pH 10. The bound phosphopeptides were fractionated from the StageTip with 50 μl of 8 buffers containing different ACN concentrations: 5%, 9%, 13%, 17%, 21%, and 80% of ACN in 200 mm NH4HCO2, pH 10. The eluted phosphopeptides were dried and stored at −20 °C. Tomato SnRK2E coding sequence was amplified from tomato cDNA with forward primer 5′-CGGAATTCATGGATCGGACGGCAGTGACA 3′ and reverse primer 5′-GCGTCGACTTACATTGCATAGACAATCTC. The PCR product was digested with EcoRI and SalI and inserted into a pGEX4T-1 vector. The construct was then introduced into BL21 cells and expressed in E. coli. Recombinant GST-SnRK2E protein was purified using glutathione-agarose beads (GST) (Sigma-Aldrich) according to the manufacturer's instructions. The in vitro kinase reaction was performed based on the siKALIP approach with some modifications (32Xue L. Wang P. Cao P. Zhu J.K. Tao W.A. Identification of extracellular signal-regulated kinase 1 (ERK1) direct substrates using stable isotope labeled kinase assay-linked phosphoproteomics.Mol. Cell. Proteomics. 2014; 13: 3199-3210Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). First, 200 μg of Lys-C digested peptides were dephosphorylated using TSAP (Roche, Basel, Switzerland) in a 1:100 (w/w) enzyme-to-peptide ratio at 37 °C overnight. The reaction was terminated by heating the sample at 75 °C for 10 min. The dephosphorylated peptides were desalted using a Sep-Pak C18 column and then suspended in kinase reaction buffer (50 mm Tris-HCl, 10 mm MgCl2, 1 mm DTT, and 1 mm γ-[18O4]-ATP, pH 7.5). The SnRK2E kinase (1 μg) was incubated with the desalted peptides at 25 °C overnight. The kinase reaction was quenched by acidifying with 10% TFA to a final concentration of 1%, and the peptides were again desalted with a Sep-Pak C18 column. The heavy 18O-phosphopeptides were further digested by trypsin at 37 °C for 6 h and enriched by PolyMAC-Ti as described above. The eluates were dried in a SpeedVac prior to LC-MS/MS analysis. The peptides were dissolved in 5 μl of 0.3% FA with 3% ACN and injected into an Easy-nLC 1000 (Thermo Fisher Scientific). Peptides were separated on a 45 cm in-house packed column (360 μm OD × 75 μm ID) containing C18 resin (2.2 μm, 100Å, (Bischoff Chromatography, Leonberg, Germany) with a column heater (Analytical Sales and Services, Flanders, New Jersey) set at 50 °C. The mobile phase buffer consisted of 0.1% FA in ultra-pure water (buffer A) with an eluting buffer of 0.1% FA in 80% ACN (buffer B) run over a linear 60 min (method comparisons) or 90 min (large-scale phosphoproteomics) gradient of 5–30% buffer B at a flow rate of 250 nL/min. The Easy-nLC 1000 was coupled online with a LTQ-Orbitrap Velos Pro mass spectrometer (Thermo Fisher Scientific). The mass spectrometer was operated in the data-dependent mode in which a full MS scan (from m/z 350–1500 with the resolution of 30,000 at m/z 400) was followed by the 10 most intense ions being subjected to collision-induced dissociation (CID) fragmentation. CID fragmentation was performed and acquired in the linear ion trap (normalized collision energy (NCE) 30%, AGC 3e4, max injection time 100 ms, isolation window 3 m/z, and dynamic exclusion 60 s). The raw files were searched directly against a Solanum lycopersicum database from the Sol Genomics Network (http://solgenomics.net) version 3.0 (33Fernandez-Pozo N. Menda N. Edwards J.D. Saha S. Tecle I.Y. Strickler S.R. Bombarely A. Fisher-York T. Pujar A. Foerster H. Yan A. Mueller L.A. The Sol Genomics Network (SGN)–from genotype to phenotype to breeding.Nucleic Acids