FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2099682053> ?p ?o ?g. }

• W2099682053 endingPage "1214" @default.
• W2099682053 startingPage "1198" @default.
• W2099682053 abstract "Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response. Perception of general elicitors by plant cells initiates signal transduction cascades that are regulated by protein phosphorylation. The earliest signaling events occur within minutes and include ion fluxes across the plasma membrane, activation of MAPKs, and the formation of reactive oxygen species. The phosphorylation events that regulate these signaling cascades are largely unknown. Here we present a mass spectrometry-based quantitative phosphoproteomics approach that identified differentially phosphorylated sites in signaling and response proteins from Arabidopsis cells treated with either flg22 or xylanase. Our approach was sensitive enough to quantitate phosphorylation on low abundance signaling proteins such as calcium-dependent protein kinases and receptor-like kinase family members. With this approach we identified one or more differentially phosphorylated sites in 76 membrane-associated proteins including a number of defense-related proteins. Our data on phosphorylation indicate a high degree of complexity at the level of post-translational modification as exemplified by the complex modification patterns of respiratory burst oxidase protein D. Furthermore the data also suggest that protein translocation and vesicle traffic are important aspects of early signaling and defense in response to general elicitors. Our study presents the largest quantitative Arabidopsis phosphoproteomics data set to date and provides a new resource that can be used to gain novel insight into plant defense signal transduction and early defense response. Plants have developed various elaborate mechanisms to ward off pathogen attack. Whereas some of these defense mechanisms are preformed and provide physical and chemical barriers to hinder pathogen infection, others are induced only upon pathogen perception. These different layers of defense include basal defense and resistance (R) 1The abbreviations used are: R, resistance; BIK1, Botrytis-Induced Kinase1; CDPK, calcium-dependent protein kinase; flg22, conserved 22-amino acid sequence of flagellin of pathogenic Pseudomonas; LTQ, linear ion trap; MAP, mitogen-activated protein; MAPK, mitogen-activated protein kinase; PM, plasma membrane; RLK, receptor-like kinase; ROI, reactive oxygen intermediate; SCX, strong cation exchange chromatography; SILAC, stable isotope labeling by amino acids in cell culture; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TiO2, titanium oxide; ABC, ATP-binding cassette; RBOHD, respiratory burst oxidase protein D. -gene-mediated defenses. Plants use R-gene products to recognize the presence or activity of specific pathogen avirulence gene products produced by specific races of microbial pathogens in a so-called gene-for-gene interaction. A specific match between an avirulence gene product and the corresponding R-gene product leads to hypersensitive response and activation of defense responses. In addition to R-gene-mediated defense plants rely on recognition of general elicitors, also called pathogen-associated molecular patterns, to detect potential pathogens and to activate basal defense (1Nurnberger T. Brunner F. Kemmerling B. Piater L. Innate immunity in plants and animals: striking similarities and obvious differences.Immunol. Rev. 2004; 198: 249-266Crossref PubMed Scopus (915) Google Scholar). These general elicitors are structural components of microbes that are recognized by plants through plasma membrane-localized receptors. The general elicitor flagellin is recognized in Arabidopsis through a conserved 22-amino acid sequence (flg22). Recognition involves the receptor-like kinase FLS2 and activates a downstream response that includes the production of reactive oxygen species, ethylene biosynthesis, activation of a MAPK cascade, and activation of defense gene expression (2Asai T. Tena G. Plotnikova J. Willmann M.R. Chiu W.L. Gomez-Gomez L. Boller T. Ausubel F.M. Sheen J. MAP kinase signalling cascade in Arabidopsis innate immunity.Nature. 2002; 415: 977-983Crossref PubMed Scopus (2041) Google Scholar, 3Gomez-Gomez L. Felix G. Boller T. A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana.Plant J. 1999; 18: 277-284Crossref PubMed Scopus (514) Google Scholar, 4Nuhse T.S. Peck S.C. Hirt H. Boller T. Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK 6.J. Biol. Chem. 2000; 275: 7521-7526Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar). Furthermore perception of flg22, as well as other general elicitors, by Arabidopsis enhances disease resistance against bacterial pathogens (5Zipfel C. Robatzek S. Navarro L. Oakeley E.J. Jones J.D. Felix G. Boller T. Bacterial disease resistance in Arabidopsis through flagellin perception.Nature. 2004; 428: 764-767Crossref PubMed Scopus (1251) Google Scholar). Perception of both avirulence gene products and general elicitors activates signal transduction cascades and early defense responses to prevent the ingression of the pathogen into host tissue. A recent study established that flg22-induced response in Arabidopsis and Avr9-induced response in tobacco show a substantial amount of overlap (6Navarro L. Zipfel C. Rowland O. Keller I. Robatzek S. Boller T. Jones J.D. The transcriptional innate immune response to flg22. Interplay and overlap with Avr gene-dependent defense responses and bacterial pathogenesis.Plant Physiol. 2004; 135: 1113-1128Crossref PubMed Scopus (454) Google Scholar). Furthermore Tao et al. (7Tao Y. Xie Z. Chen W. Glazebrook J. Chang H.S. Han B. Zhu T. Zou G. Katagiri F. Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae.Plant Cell. 2003; 15: 317-330Crossref PubMed Scopus (590) Google Scholar) have shown that the transcriptional reprogramming in response to compatible and incompatible interactions is very similar and that the difference in outcome likely depends on the quantitative nature of the local defense response (7Tao Y. Xie Z. Chen W. Glazebrook J. Chang H.S. Han B. Zhu T. Zou G. Katagiri F. Quantitative nature of Arabidopsis responses during compatible and incompatible interactions with the bacterial pathogen Pseudomonas syringae.Plant Cell. 2003; 15: 317-330Crossref PubMed Scopus (590) Google Scholar). The signal transduction pathways activated by general elicitors and Avrgene products are not well characterized. Thus far, research on plant defense signaling has focused mainly on the genetic dissection of the signal transduction pathways. Although the analysis of mutants with altered defense responses has yielded a variety of putative defense signaling components, the molecular mechanisms these signaling components affect remain elusive. However, reverse and forward genetic approaches have pointed to an important role for protein phosphorylation in defense signaling. Mutations in the Pto serine/threonine kinase in tomato (8Martin G.B. Brommonschenkel S.H. Chunwongse J. Frary A. Ganal M.W. Spivey R. Wu T. Earle E.D. Tanksley S.D. Map-based cloning of a protein kinase gene conferring disease resistance in tomato.Science. 1993; 262: 1432-1436Crossref PubMed Scopus (1118) Google Scholar) and Xa21 leucine-rich repeat kinase in rice (9Song W.Y. Wang G.L. Chen L.L. Kim H.S. Pi L.Y. Holsten T. Gardner J. Wang B. Zhai W.X. Zhu L.H. Fauquet C. Ronald P. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21.Science. 1995; 270: 1804-1806Crossref PubMed Scopus (1772) Google Scholar) compromise the plant's resistance to race-specific pathogens, whereas mutations in receptor-like kinase FLS2 in Arabidopsis enhances susceptibility to Pseudomonas syringae pv. tomato DC3000 (5Zipfel C. Robatzek S. Navarro L. Oakeley E.J. Jones J.D. Felix G. Boller T. Bacterial disease resistance in Arabidopsis through flagellin perception.Nature. 2004; 428: 764-767Crossref PubMed Scopus (1251) Google Scholar). Silencing of tobacco MAPK kinase kinase NPK1 and Arabidopsis MAPK MPK6 also compromises disease resistance (10Jin H. Axtell M.J. Dahlbeck D. Ekwenna O. Zhang S. Staskawicz B. Baker B. NPK1, an MEKK1-like mitogen-activated protein kinase kinase kinase, regulates innate immunity and development in plants.Dev. Cell. 2002; 3: 291-297Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 11Menke F.L. van Pelt J.A. Pieterse C.M. Klessig D.F. Silencing of the mitogen-activated protein kinase MPK6 compromises disease resistance in Arabidopsis.Plant Cell. 2004; 16: 897-907Crossref PubMed Scopus (182) Google Scholar). The perception of general elicitors and downstream signal transduction has been studied intensively with biochemical and pharmacological approaches and more recently with transient expression approaches in protoplasts (2Asai T. Tena G. Plotnikova J. Willmann M.R. Chiu W.L. Gomez-Gomez L. Boller T. Ausubel F.M. Sheen J. MAP kinase signalling cascade in Arabidopsis innate immunity.Nature. 2002; 415: 977-983Crossref PubMed Scopus (2041) Google Scholar, 12Felix G. Duran J.D. Volko S. Boller T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin.Plant J. 1999; 18: 265-276Crossref PubMed Scopus (1153) Google Scholar, 13Menke F.L. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in Catharanthus roseus.Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar, 14Chivasa S. Hamilton J.M. Pringle R.S. Ndimba B.K. Simon W.J. Lindsey K. Slabas A.R. Proteomic analysis of differentially expressed proteins in fungal elicitor-treated Arabidopsis cell cultures.J. Exp. Bot. 2006; 57: 1553-1562Crossref PubMed Scopus (94) Google Scholar, 15Ndimba B.K. Chivasa S. Hamilton J.M. Simon W.J. Slabas A.R. Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors.Proteomics. 2003; 3: 1047-1059Crossref PubMed Scopus (134) Google Scholar, 16Bindschedler L.V. Dewdney J. Blee K.A. Stone J.M. Asai T. Plotnikov J. Denoux C. Hayes T. Gerrish C. Davies D.R. Ausubel F.M. Paul Bolwell G. Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance.Plant J. 2006; 47: 851-863Crossref PubMed Scopus (420) Google Scholar). This has led to the identification of several early signaling events, including ion fluxes across the plasma membrane, extracellular alkalinization, transient increases in cytosolic calcium concentration, protein phosphorylation and dephosphorylation through activation of MAPK and calcium-dependent protein kinases (CDPKs), and the production of extracellular reactive oxygen species through PM-localized NADPH oxidase and apoplastic peroxidase and biosynthesis of ethylene and jasmonic acid (13Menke F.L. Parchmann S. Mueller M.J. Kijne J.W. Memelink J. Involvement of the octadecanoid pathway and protein phosphorylation in fungal elicitor-induced expression of terpenoid indole alkaloid biosynthetic genes in Catharanthus roseus.Plant Physiol. 1999; 119: 1289-1296Crossref PubMed Scopus (214) Google Scholar, 16Bindschedler L.V. Dewdney J. Blee K.A. Stone J.M. Asai T. Plotnikov J. Denoux C. Hayes T. Gerrish C. Davies D.R. Ausubel F.M. Paul Bolwell G. Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance.Plant J. 2006; 47: 851-863Crossref PubMed Scopus (420) Google Scholar, 17Scheel D. Resistance response physiology and signal transduction.Curr. Opin. Plant Biol. 1998; 1: 305-310Crossref PubMed Scopus (225) Google Scholar). Eventually both receptor-mediated perception of general elicitors and R-gene-mediated detection of avirulence proteins lead to the activation of transcriptional cascades and the expression of defense responses. By definition, the proteins that make up the signal transduction pathway are present in the cell prior to the perception of the elicitor. They are activated by post-translational modifications, conformational changes, and/or changes in complex formation. The most widely recognized post-translational modification involved in signal transduction is protein phosphorylation. Although protein kinases and phosphatase have been identified with a role in defense signaling and resistance, their exact role and their substrates have remained elusive in plants. General elicitor signaling is thought to be initiated through interaction of the elicitor with a plasma membrane-localized receptor. Immediate early signaling and responses are likely mediated through plasma membrane-associated proteins. Recent work showed that many membrane-associated proteins have one or more phosphorylation sites (18Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (411) Google Scholar). Furthermore phosphorylated proteins have been detected in the extracellular matrix of Arabidopsis suspension cultured cells in response to fungal elicitor (15Ndimba B.K. Chivasa S. Hamilton J.M. Simon W.J. Slabas A.R. Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors.Proteomics. 2003; 3: 1047-1059Crossref PubMed Scopus (134) Google Scholar). However, the functionality of only a very few of these is currently understood. The large scale identification of phosphorylation sites as well as the kinetics and stoichiometry of the phosphorylation status will help unravel the immediate early signal transduction cascades and defense response in response to general elicitors. Detection of changes in protein phosphorylation was traditionally performed by labeling cells with radioactive phosphate followed by two-dimensional gel separation of labeled proteins and mass spectrometric analysis (19Peck S.C. Nuhse T.S. Hess D. Iglesias A. Meins F. Boller T. Directed proteomics identifies a plant-specific protein rapidly phosphorylated in response to bacterial and fungal elicitors.Plant Cell. 2001; 13: 1467-1475Crossref PubMed Scopus (188) Google Scholar). Recent advances in LC-MS/MS have made it possible to directly analyze highly complex protein mixtures. Unfortunately traditional LC-MS/MS experiments generally yield few phosphorylated peptides. Despite the fact that a high percentage of cellular proteins are thought to be phosphorylated at any given time (20Hunter T. The Croonian Lecture 1997. The phosphorylation of proteins on tyrosine: its role in cell growth and disease.Philos. Trans. R. Soc. Lond. B Biol. Sci. 1998; 353: 583-605Crossref PubMed Scopus (364) Google Scholar), this phosphorylated form is generally low in abundance. Furthermore the addition of a phosphate group often results in a reduction in ionization efficiency in MS (21Steen H. Jebanathirajah J.A. Rush J. Morrice N. Kirschner M.W. Phosphorylation analysis by mass spectrometry: myths, facts, and the consequences for qualitative and quantitative measurements.Mol. Cell. Proteomics. 2006; 5: 172-181Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Recent advances in selective purification of phosphopeptides have made it possible to overcome these limitations and identify phosphorylation sites in large numbers of proteins (18Nuhse T.S. Stensballe A. Jensen O.N. Peck S.C. Phosphoproteomics of the Arabidopsis plasma membrane and a new phosphorylation site database.Plant Cell. 2004; 16: 2394-2405Crossref PubMed Scopus (411) Google Scholar, 22Ficarro S.B. McCleland M.L. Stukenberg P.T. Burke D.J. Ross M.M. Shabanowitz J. Hunt D.F. White F.M. Phosphoproteome analysis by mass spectrometry and its application to Saccharomyces cerevisiae.Nat. Biotechnol. 2002; 20: 301-305Crossref PubMed Scopus (1499) Google Scholar, 23Pinkse M.W. Uitto P.M. Hilhorst M.J. Ooms B. Heck A.J. Selective isolation at the femtomole level of phosphopeptides from proteolytic digests using 2D-NanoLC-ESI-MS/MS and titanium oxide precolumns.Anal. Chem. 2004; 76: 3935-3943Crossref PubMed Scopus (830) Google Scholar). Although these qualitative approaches are highly informative in themselves, the identification of phosphorylation events relevant to a specific signaling pathway requires quantitative rather than qualitative measurements. Quantitative phosphoproteomics approaches based on stable isotope labeling by amino acids in cell culture (SILAC) have been successfully applied in studies with cultured human cells and in yeast (24Ballif B.A. Roux P.P. Gerber S.A. MacKeigan J.P. Blenis J. Gygi S.P. Quantitative phosphorylation profiling of the ERK/p90 ribosomal S6 kinase-signaling cassette and its targets, the tuberous sclerosis tumor suppressors.Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 667-672Crossref PubMed Scopus (182) Google Scholar, 25Blagoev B. Ong S.E. Kratchmarova I. Mann M. Temporal analysis of phosphotyrosine-dependent signaling networks by quantitative proteomics.Nat. Biotechnol. 2004; 22: 1139-1145Crossref PubMed Scopus (589) Google Scholar, 26Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar) and could in principle also be used in studies with plant cell cultures. An alternative approach, called iTRAQ (isobaric tagging for relative and absolute quantitation), has been described recently in several quantitative phosphoproteomics studies (27Kim J.E. White F.M. Quantitative analysis of phosphotyrosine signaling networks triggered by CD3 and CD28 costimulation in Jurkat cells.J. Immunol. 2006; 176: 2833-2843Crossref PubMed Scopus (93) Google Scholar, 28Sachon E. Mohammed S. Bache N. Jensen O.N. Phosphopeptide quantitation using amine-reactive isobaric tagging reagents and tandem mass spectrometry: application to proteins isolated by gel electrophoresis.Rapid Commun. Mass Spectrom. 2006; 20: 1127-1134Crossref PubMed Scopus (51) Google Scholar) and is based on the labeling of the N termini and lysine residues of extracted (phospho-)peptides with isobaric tags to allow quantitation. Here we report an alternative quantitative phosphoproteomics approach, based on 14N/15N metabolic labeling, that is specifically suited for cultured plant cells. We set up this quantitative phosphoproteomics approach to identify the early signaling and response proteins modulated upon perception of general elicitors by Arabidopsis cells. We used a 14N/15N metabolic labeling strategy that allowed us to directly compare phosphorylation levels of proteins of mock-treated and elicitor-treated cells with mass spectrometry. Changes in phosphorylation of membrane-associated proteins were analyzed for cells treated with the flagellin peptide flg22 and fungal elicitor xylanase. Quantitative changes in phosphorylation were compared between the two resulting data sets so that proteins phosphorylated in response to both elicitors could be identified. Our data identify quantitative changes in phosphorylation of novel membrane-associated proteins as well as established defense-related proteins. Additionally our data also point to a role for protein translocation and vesicle transport during early elicitor signaling and response. Poros R3 was obtained from Applied Biosystems (Nieuwerkerk a/d lJssel, The Netherlands). K15NO3 and (15NH4)2SO4 were from Spectra Stable Isotopes (Columbia, MD). Flagellin peptide flg22 was synthesized by Sigma Genosys. Modified trypsin was from Roche Applied Science. GeLoader tips came from Eppendorf (Eppendorf, Germany). The Empore C8 disk was from 3M (Neuss, Germany). Syringes and needles were from SGE (Victoria, Australia). All other chemicals were obtained from Sigma-Aldrich. Suspension cells of Arabidopsis thaliana accession Col-0 (29Axelos M. Curie C. Mazzolini L. Bardet C. Lescure B. A protocol for transient gene expression in Arabidopsis thaliana protoplasts isolated from cell suspension cultures.Plant Physiol. Biochem. 1992; 30: 123-128Google Scholar) were cultured on Gamborg B5 medium (30 g/liter sucrose, 1 mm naphthaleneacetic acid, 1.0 mm (NH4)2SO4, 25 mm KNO3, 1.1 mm NaH2PO4·H2O, 1.0 mm CaCl2·2H2O, 1.0 mm MgSO4·7H2O, 100 μm FeNaEDTA, 60 μm MnSO4·H2O, 50 μm H3BO3, 7.0 μm ZnSO4, 1.0 μm Na2MoO4, 4.5 μm KI, 0.10 μm CoCl2, 0.10 μm CuSO4, and 112 mg/ml vitamin mixture (G0415, Duchefa, Haarlem, The Netherlands)) in 50-ml cultures in 250-ml flasks with shaking at 150 rpm. Growth conditions were 8 h of light of 70 μm m−2 s−1 photosynthetic photon flux density at 25 °C. Cells were subcultured (4 ml in 50 ml) every 7 days. Experiments were performed 5 days after subculturing. Cultures were grown for at least 4 subculture periods of 7 days on either 14N- or 15N-medium prior to elicitor induction. Four-day-old cultures were treated for exactly 10 min with 100 μg/ml xylanase or 1 μm flg22. For the first biological replicate, three separately treated (14N) cultures and three mock-treated (15N) cultures were pooled, harvested by filtration, washed with ice-cold 20 mm KNO3, and resuspended in homogenization buffer (100 mm HEPES, 330 mm sucrose, 10 mm Na2EDTA, 10 mm EGTA, 50 mm sodium pyrophosphate, 25 mm NaF, 1 mm NaMoO4, 1 mm MgCl2, 5 mm DTT, 1 mm ascorbic acid, 1 mm Na3VO4, 5 g/liter polyvinylpolypyrrolidone, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml aprotinin) at a ratio of 2 ml/g of cells. This was done seven times for a total of 21 cultures of each treatment type per experiment, and the homogenates were pooled before PM isolation. In the second biological replicate 15N-grown cultures were elicitor-treated, and 14N-grown cultures were mock-treated. Both elicitor treatments and both biological replicas were done independently with a 7-day period between each treatment and each replica. Cells were broken in a 60-ml Potter-Elvehjem homogenizer on ice (8 min at 1000 rpm). The homogenate was centrifuged for 10 min at 10,000 × g (Sorvall SW34 rotor). The supernatant was centrifuged for 1 h at 100,000 × g (Beckman SW28 rotor). The pellet was resuspended in a buffer containing 330 mm sucrose, 5 mm KPO4, pH 7.8, and 5 mm KCl and subjected to aqueous two-phase partitioning (30Larsson C. Sommarin M. Widell S. Isolation of highly purified plant plasma membranes and separation of inside-out and right-side-out vesicles.Methods Enzymol. 1994; 228: 451-469Crossref Scopus (203) Google Scholar) using 6.4% (w/w) each of dextran T500 and polyethylene glycol 3350 with 5 mm KCl and 5 mm KPO4, pH 7.8. The U3 phase was diluted 2-fold in buffer containing 330 mm sucrose and 5 mm KPO4, pH 7.8. Plasma membranes were pelleted at 100,000 × g for 1 h (Beckman SW28 rotor), resuspended to a concentration of 10 μg/μl, frozen in liquid nitrogen, and stored at −80 °C. Plasma membranes containing 4 mg of protein (50% of the total isolate) were mixed with a solution containing 2% (w/v) Brij58 in 330 mm sucrose and 5 mm KPO4 with inhibitors (1 mm NaVO3, 1 mm NaMoO4, 25 mm NaF, 1 μg/μl leupeptin, 1 μg/μl aprotinin, 1 μg/μl pepstatin, and 1 mm PMSF) to a detergent:protein ratio of 3:1 (w/w). After centrifugation (200,000 × g for 1 h) the pellet was resuspended in 200 mm KCl and 30 mm DTT with inhibitors and incubated for 2 h at room temperature. This was centrifuged again, and the pellet was resuspended in 50 mm iodoacetamide in 50 mm NH4HCO3 and kept at room temperature for 30 min in darkness. One more wash with 50 mm NH4HCO3 was performed, and the final pellet was resuspended in 250 μl of 50 mm NH4HCO3. Proteins were digested with trypsin (ratio, 1:50) overnight at 37 °C. The solution was acidified with formic acid (5% end concentration) and centrifuged as above. The supernatant was then brought to a final volume of 40 μl under reduced pressure. Strong cation exchange chromatography (SCX) was performed according to Gruhler et al. (26Gruhler A. Olsen J.V. Mohammed S. Mortensen P. Faergeman N.J. Mann M. Jensen O.N. Quantitative phosphoproteomics applied to the yeast pheromone signaling pathway.Mol. Cell. Proteomics. 2005; 4: 310-327Abstract Full Text Full Text PDF PubMed Scopus (698) Google Scholar) on an ammonium formate gradient. Prior to SCX the digested peptides were desalted on a 15-μl POROS R3 reverse phase column made in a constricted GeLoader tip (31Gobom J. Nordhoff E. Mirgorodskaya E. Ekman R. Roepstorff P. Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry.J. Mass Spectrom. 1999; 34: 105-116Crossref PubMed Scopus (631) Google Scholar). Peptides were eluted with 40 μl of 50% acetonitrile and diluted to yield 100 μl with SCX buffer A. Peptides were separated on a 1-ml Resource S column (GE Healthcare) on an ÁKTA purifier (GE Healthcare). 16 fractions were collected, reduced in volume to 20 μl, and frozen at −20 °C. Titanium oxide (GL Sciences Inc., Tokyo, Japan) columns were prepared by placing a small plug, stamped out of a 3M Empore C8 disk using a 0.3-mm-inner diameter flat tip needle fitted on a 1-ml glass syringe and brought to position using a 0.15-mm-outer diameter wire just above the constricted end of a GeLoader tip. The column material was then packed to a bedding volume of 1 μl (±2 cm) and washed with washing buffer (80% acetonitrile in 0.1% TFA). Samples were loaded on a 3-μl reverse phase column as described above and eluted with 15 μl of titanium oxide (TiO2) loading buffer (80% acetonitrile, 0.1% TFA, and 300 mg/ml dihydroxybenzoic acid). This eluate was loaded slowly onto the TiO2 column and washed with 6 μl of loading buffer and 6 μl of washing buffer. Phosphopeptides were eluted with 20 μl of 1% ammonia. The eluate was mixed with 20 μl of 10% formic acid and frozen at −20 °C prior to MS analysis. Nanoscale HPLC-MS/MS experiments were performed on an Agilent 1100 nanoflow system (Agilent Technologies) connected to a 7-tesla Finnigan LTQ-FT mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a nanoelectrospray ion source. Loading was accomplished by using a flow rate of 5 μl/min onto a homemade 2-cm fused silica precolumn (100-μm inner diameter, 375-μm outer diameter, Resprosil C18-AQ, 3 μm (Maisch, Ammerbuch, Germany)) using an autosampler. Sequential elution of peptides was accomplished using a linear gradient from Solution A (0.6% acetic acid) to 50% Solution B (80% acetonitrile and 0.5% acetic acid) in 40 min over the precolumn in line with a homemade 20–25-cm resolving column (50-μm inner diameter, 375-μm outer diameter, Resprosil C18-AQ, 3 μm (Maisch)). The mass spectrometer was operated in the data-dependent mode to automatically switch between MS, MS/MS, and neutral loss-dependent MS/MS/MS (MS3) acquisition. Survey full-scan MS spectra (from m/z 300 to 1500) were acquired in the FT-ICR mass spectrometer with resolution R = 25,000 at m/z 400 (after accumulation to a target value of 5,000,000 in the linear ion trap). The three most intense ions were sequentially isolated for accurate mass measurements by a FT-ICR “single ion monitoring scan,” which consisted of a 15-Da mass range, resolution R = 50,000, and target accumulation value of 80,000. These were then simultaneously fragmented in the linear ion trap using collisionally induced dissociation at a target value of 10,000. The data-dependent neutral loss algorithm in the Xcalibur software was enabled for each MS/MS spectrum. Data-dependent settings were chosen to trigger a MS3 scan when a neutral loss corresponding to 98, 49, or 32.7 Th was detected among the five most intense fragment ions. Former target ions selected for MS/MS were dynamically excluded for 30 s. Total cycle time was approximately 3 s. Each sample was also subjected to a “regular” LC-MS/MS analysis. Survey full-scan MS spectra (from m/z 300 to 1500) were acquired in the FT-ICR mass spectrometer with resolution R = 100,000 at m/z 400 (after accumulation to a target value of 2,000,000 in the linear ion trap). The two most intense ions were fragmented in the linear ion trap using collisionally induced dissociation at a target value of 10,000. All MS/MS spectra files from each LC ru" @default.
• W2099682053 created "2016-06-24" @default.
• W2099682053 creator A5015833446 @default.
• W2099682053 creator A5037662438 @default.
• W2099682053 creator A5037845155 @default.
• W2099682053 creator A5040324996 @default.
• W2099682053 creator A5053603496 @default.
• W2099682053 creator A5071143347 @default.
• W2099682053 date "2007-07-01" @default.
• W2099682053 modified "2023-10-11" @default.
• W2099682053 title "Quantitative Phosphoproteomics of Early Elicitor Signaling in Arabidopsis" @default.
• W2099682053 cites W1495926848 @default.
• W2099682053 cites W1581562089 @default.
• W2099682053 cites W1826812957 @default.
• W2099682053 cites W1918775481 @default.
• W2099682053 cites W1968234761 @default.
• W2099682053 cites W1972004632 @default.
• W2099682053 cites W1975938671 @default.
• W2099682053 cites W1978254752 @default.
• W2099682053 cites W1978893949 @default.
• W2099682053 cites W1979456040 @default.
• W2099682053 cites W1984017573 @default.
• W2099682053 cites W1993480266 @default.
• W2099682053 cites W1999954691 @default.
• W2099682053 cites W2004704197 @default.
• W2099682053 cites W2011820514 @default.
• W2099682053 cites W2028254759 @default.
• W2099682053 cites W2033889320 @default.
• W2099682053 cites W2040400491 @default.
• W2099682053 cites W2043581879 @default.
• W2099682053 cites W2059555945 @default.
• W2099682053 cites W2059971395 @default.
• W2099682053 cites W2064603411 @default.
• W2099682053 cites W2065873936 @default.
• W2099682053 cites W2066036610 @default.
• W2099682053 cites W2071507590 @default.
• W2099682053 cites W2071555141 @default.
• W2099682053 cites W2072425598 @default.
• W2099682053 cites W2073376866 @default.
• W2099682053 cites W2074512572 @default.
• W2099682053 cites W2076035297 @default.
• W2099682053 cites W2077200333 @default.
• W2099682053 cites W2097558464 @default.
• W2099682053 cites W2097847369 @default.
• W2099682053 cites W2099255819 @default.
• W2099682053 cites W2099374185 @default.
• W2099682053 cites W2102340487 @default.
• W2099682053 cites W2108253576 @default.
• W2099682053 cites W2111547922 @default.
• W2099682053 cites W2112142256 @default.
• W2099682053 cites W2112351234 @default.
• W2099682053 cites W2115177316 @default.
• W2099682053 cites W2116754759 @default.
• W2099682053 cites W2117396466 @default.
• W2099682053 cites W2123208777 @default.
• W2099682053 cites W2123844599 @default.
• W2099682053 cites W2128640142 @default.
• W2099682053 cites W2128987420 @default.
• W2099682053 cites W2129777487 @default.
• W2099682053 cites W2140946052 @default.
• W2099682053 cites W2146884230 @default.
• W2099682053 cites W2147757443 @default.
• W2099682053 cites W2147863419 @default.
• W2099682053 cites W2149118179 @default.
• W2099682053 cites W2153422220 @default.
• W2099682053 cites W2153777219 @default.
• W2099682053 cites W2155880596 @default.
• W2099682053 cites W2157766441 @default.
• W2099682053 cites W2160504799 @default.
• W2099682053 cites W2160913295 @default.
• W2099682053 cites W2163423253 @default.
• W2099682053 cites W2166793037 @default.
• W2099682053 cites W2169587278 @default.
• W2099682053 cites W2170177782 @default.
• W2099682053 cites W2171320885 @default.
• W2099682053 cites W2215682328 @default.
• W2099682053 cites W4232051501 @default.
• W2099682053 cites W65131259 @default.
• W2099682053 doi "https://doi.org/10.1074/mcp.m600429-mcp200" @default.
• W2099682053 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/17317660" @default.
• W2099682053 hasPublicationYear "2007" @default.
• W2099682053 type Work @default.
• W2099682053 sameAs 2099682053 @default.
• W2099682053 citedByCount "616" @default.
• W2099682053 countsByYear W20996820532012 @default.
• W2099682053 countsByYear W20996820532013 @default.
• W2099682053 countsByYear W20996820532014 @default.
• W2099682053 countsByYear W20996820532015 @default.
• W2099682053 countsByYear W20996820532016 @default.
• W2099682053 countsByYear W20996820532017 @default.
• W2099682053 countsByYear W20996820532018 @default.
• W2099682053 countsByYear W20996820532019 @default.
• W2099682053 countsByYear W20996820532020 @default.
• W2099682053 countsByYear W20996820532021 @default.
• W2099682053 countsByYear W20996820532022 @default.
• W2099682053 countsByYear W20996820532023 @default.
• W2099682053 crossrefType "journal-article" @default.
• W2099682053 hasAuthorship W2099682053A5015833446 @default.

• next