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For Xoo, peptone sucrose (PS) medium (peptone 10 gL-1, sucrose 10 gL-1, L-glutamic acids 1 gL-1) and XOM2 were used as rich and minimal media, respectively. For Xcv and Xag, tryptic soy (TS) medium (Tryptic Soy Broth Soybean-Casein Digested 30 gL-1). tryptic soy (TS) medium (Tryptic Soy Broth Soybean-Casein Digested 30g/L). In minimal media, XOM2 was used for XOO and XVM2 was used for XCV and Xag. Protein extraction and Peptide preparation Bacteria cells grown to 0.6 at OD600 were harvested by centrifugation (15 min, 7,300 x g, 28 oC). The 0.5 g (wet weight) of cells were resuspended in 1 ml of 50mM Tris-HCl (pH7.8) and centrifuged again (10 min, 7,300 x g, 28 oC). This washing process was repeated two times. After removing the supernatants, the pellets were resuspended in 1ml of lysis buffer (6 M Guanidine-HCl, 10mM DTT, 50 mM Tris-HCl, pH 7.8). Resuspended cells were disrupted by Ultrasonic Processor (Cole Parmer) ten times (10 s on / 60 s off cycle) in ice, and centrifuged (20 min, 8,000 x g, 4 oC). After collecting supernatants, concentration of proteins was quantified by BCA protein assay kit (ThermoFisher) and 1,000 μg of samples were used for the next step. Samples were incubated for 1 hr at 60℃, alkylated by treatment of 100mM iodoacetamide in the dark chamber at room temperature for 1 hr, and then incubated again with 20 mM DDT (final concentration) at room temperature for 1hr. For protein precipitation, 0.3 volumes of ice-cold trichloroacetic acid were added into the samples, and incubated at 4oC for 12 hrs. After centrifugation (30 min, 15,700 x g, 4 oC), pellets were resuspended in 1ml of acetone (Sigma), and centrifuged again (30 min, 15,700 x g, 4 oC). This process was repeated three times. The pellets were dried in clean bench and dissolved in the start volume of 50mM ammonium bicarbonate (pH 7.8). For digestion of proteins, after 5 μg of trypsin (Promega) were treated in 200 μg of extracted proteins, the mixture were incubated for 24 hrs at 37 oC. Trypsin-treated samples were acidified with 0.4% trifluoroacetic acid (TFA, Sigma), and loaded into the Sep-Pak Vac 1cc tC18 cartridges (Waters) for cleaning samples. After washing the cartridges by 1 ml of 0.1% TFA, samples were eluted with 1 ml of elution buffers (0.1% TFA, 50% acetonitrile). Eluted samples were dried by Speed Vac concentrator (Vision), dissolved in 30 μl of 0.4% acetic acid, and quantified the protein by BCA protein assay kit (ThermoFisher). Samples were diluted to 1 μg/μl for mass spectrometry analysis.Prideiodoacetamide derivatized residueacetylated residueSpectrum countingMass spectrometry analysis Tryptic peptide mixtures from each sample (2 μl, ~ 2 μg) were analyzed by split free nano LC (EASY-nLC II, ThermoFisher) connected to LTQ Velos Pro instrument (ThermoFisher) via a nanospray ion mode. Sample digested were separated by a column packed in-house with 7.5 cm of MAGIG C18AQ 200A (5 μm) material (Michrom). Peptides were eluted over a 420 min gradient at a flow rate of 300 nL/min by a water/ACN gradient (Solvent A, Water with 0.1% Formic Acid; Solvent B, 100% Acetonitrile with 0.1% Formic Acid) consisting of 5 min 7% B, 380 min gradient to 35% B, and 10 min to 80% B with final hold at 7% B for 25 min. Full MS spectra were acquired in 6 data dependent MS/MS scans over a mass range of m/z 300–2000. Dynamic exclusion was allowed with a repeat count of 1, repeat duration of 0.5 min, and exclusion duration of 3.0 min, with charge state selection enable to preferentially select 2+ and 3+ ions. Peptides were transferred with a 1.8 kV spray voltage and a desolvation capillary temperature of 200 oC. Up to the 6 most intense ions in each full MS scan were consecutively collected for fragmentation and examined in centroid mode within the linear ion trap part of the instrument. Three biological replicates were carried out. Protein/Peptide identification and quantification Thermo Proteome Discoverer 1.3(ver. 1.3.0.399) with SEQUEST search algorithm was used for interpretation of acquired MS/MS spectra. Spectra were searched against the Xcv strain 85-10 database. Trypsin was fixed as an enzyme, and two missed cleavage was allowed. All accepted Xcv peptides had a false discovery rate (FDR) of 0.01 with reversed database searches, and precursor mass accuracies of 100 ppm. In addition, probability scores of all peptides were >20.Integrated decoy database search were carried out. The oxidation of methionine was fixed as a possible modification. Proteins matched at least two unique peptides were considered as present in the sample. And then, proteins identified were re-imported into scaffold 4 proteome software for visualization of proteomics data and comparison between biological samples. For comparative analyses, each protein were normalized with total peptide spectra matches (PSM). The average of normalized PSMs was calculated per protein and used as a comparison value to identify differently expressed proteins between xanthomonomas spp. in rich-media and minimal-media, respectively.ProteomicsSang-Wook HanLTQPARTIALDept. Integrative Plant Science, Chuang-Ang UniversityXanthomonas Campestris Pv. Vesicatoria Str. 85-10Xanthomonas Oryzae Pv. Oryzae (strain Pxo99a)Xanthomonas Axonopodis Pv. Glycines Str. 8ra29044975 Park HJ, Bae N, Park H, Kim DW, Han SW. Comparative proteomic analysis of three Xanthomonas spp. cultured in minimal and rich media. Proteomics. 2017 10.1002/pmic.201700142swhan@cau.ac.krBiologicalChung-Ang UniversityKorea, Republic ofBacteria change their gene expression when exposed to different nutrient conditions. The levels of proteins do not always correlate with those of RNAs, hence proteomic analysis is required for understanding how bacteria adapt to different conditions. Herein, differentially abundant proteins from Xanthomonas oryzae pv. oryzae (Xoo), X. campestris pv. vesicatoria (Xcv), and X. axonopodis pv. glycines (Xag), which were cultured in rich media and in minimal media, were determined using label-free shotgun proteomic analysis and clusters of orthologous groups classification. The detected proteins from all three species ranged from 1190 to 1187. Among them, 702, 584, and 529 proteins from Xoo, Xcv, and Xag, respectively, were more (> twofold) abundant depending on the media, indicating that about 11.4-13.8% of proteins from the three species were differentially expressed. The levels of abundant proteins in minimal media were significantly higher than those in rich media for all three species, demonstrating how Xanthomonas species actively change their protein expression in different nutrient conditions. These results will lead to new insights in elucidation of cellular mechanisms involved in virulence and adaption of bacteria to harsh environments for further studies. The MS proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD006310.Comparative Proteomic Analysis of Three Xanthomonas spp. Cultured in Minimal and Rich Media.Park Hye-Jee HJ, Bae Nahee N, Park Hanbi H, Kim Dae-Wi DW, Han Sang-Wook SWprojections, ZFYVE8, IL1BC, MeCN, PLXN5, Vinegar, fond, DmelCG2903, CASP-1, l(2)23AB5, growth and development, chronic obstructive pulmonary disease (COPD), CycEI, Apaf-1, trichloro-, SeP, Bca, 2-Propanone, Trifluoroacetate, Polypeptides, protein polypeptide chains, KL receptor activity, hydrogen chloride, CEH, E260, T22F8_160, sense of sight, GRP1, ATGPR7, CH3-C#N, Grp1, Vps27, SCO5, Chronic irreversible airway obstruction, HCL-C, 1, l(2)23Ad(Hrs), 2, GRP8, NEPII, AGM4, GRP7, SCO1, 4, SEH, Il1bc, Gsfsow3, Sep, SEP, bca, hac-1, PTPSTEP, CAFL - Chronic airflow limitation, proteins, W, Acetic Acid Glacial, ecotype, IL-1BC, sEP, AI047805, Wasserstoffchlorid, Bacteria <bacteria>, disease (COPD), Prokaryotae, MeCO2H, Bs, Procaryotae, autolysin activity, GPH, chronic obstructive lung disease [Ambiguous], anatomical protrusion, Caspase-1 subunit p10, HRS, Hrs, Chp, anon-53Fa, l(2)SH0173, Guanidine Monohydroiodine, SRp40, Cyc E, 4' Dichlorodiphenyltrichloroethane, DmelCG7788, CHRONIC OBSTRUCTIVE AIRWAY DIS, PBT, TbisC-ethane, Glacial, BG:DS07108.3, hrs, Dm1, shelf, Chlorwasserstoff, vision, prokaryotes, SRP40, leukemic reticuloendotheliosis, l(2)05206, Airflow Obstruction, CG6829, dark/hac-1/dapaf-1, GLYCINE-RICH PROTEIN 8, Chronic Obstructive Airways Disease, Acid, pbt, Chronic obstructive lung disease, Ethylic acid, CG11628, LY57, alpha-D-Glucopyranoside, Guanidine Phosphate, Striatum-enriched protein-tyrosine phosphatase, projection, beta Trypsin, ridge, F2G1.4, OBSTRUCTIVE PULMONARY DISEASE (COPD), Acide, Drice, DDCT, bacteriolytic toxin activity, Benzene, methyl cyanide, chlorane, DRICE, Prokaryota, krk1, Benzochloryl, Sucrose, C78655, Acide trichloracetique, cycline, GRP1/cytohesin 1, chlorure d'hydrogene, lamellae, DmcyclinE, DrIce, DrICE, CAO - Chronic airflow obstruction, beta-D-Fruf-(2<->1)-alpha-D-Glcp, Glutamic Acids, CHRONIC OBSTRUCTIVE PULM DIS, Chronic obstructive pulmonary disease NOS, protein-containing complex, process of organ, CG7826, and RNA binding 1, cdi7, CG11633, Chronic obstructive pulmonary disease finding, lamella, Glutamic Acid Derivatives, PULMONARY DISEASE (COPD), Guanidium, cyclinE, Ly57, CG7835, Cdi7, CDI7, Gene Products, CG42273, trichloracetique, Cesium Trifluoroacetate, Monohydroiodine, Hydrogen chloride, soybeans, PCE-2, beta-Trypsin, DmcycE, soybean, Monohydrobromide, Guanidinium Chloride, hairy cell leukemia, GL-1, Caspase-1 subunit p20, ATGRP7, ATGRP8, 2-bis(p-chlorophenyl)ethane, NEC, Chronic, peptidos, SUCROSE, laminae, proto-oncogene c-Kit, dark/dapaf-1/hac-1, bacteria, Guanidine Monohydrate, AcOH, Guanidine Nitrate, 1-(14)C-labeled, chronic obstructive airways disease NOS, caspase 3, USH3B, anatomical process, dApaf-1, Proteins, COAD, DRPLA, stepk, Mell1, chloridohydrogen, CE-2, Mischung, COLD - Chronic obstructive lung disease, acetonitrile, buffer, Cell, chronic, polypeptide, APAF1, l(2)k02514, sacarosa, native protein, DmCycE, Temperatures, Hac-1, KIT ligand receptor activity, Strain, Nitrate, Sodium, (COPD), 1-trichloro-2, p', chronic airway obstruction, chronic obstructive, CAL - Chronic airflow limitation, organ process, XKrk1, AU020952, Guanidine Monohydrobromide, Guanidine, holin, l(2)k02602, Guanidine Sulfate, postnatal growth, GR-RBP7, GR-RBP8, Chloride, Protein pp110, Dark/Dapaf-1/HAC1, IL-1 beta-converting enzyme, ethanenitrile, Interleukin-1 beta-converting enzyme, CD117, DmelCG11628, Glutaminic Acids, ME-IV, processes, D-CycE, Gene Proteins, C-Kit, MeCOOH, ACETIC ACID, prokaryote, Ssm, Glycine max, chronic obstructive lung disease (disorder), xkl-1, Sulfate, hydrochloric acid, NEP2, Ethanoic acid, chronic obstructive pulmonary disease, Sfrs5, Hac-1/Dark, TbisC ethane, T22F8.160, NCMe, and rna binding 2, DmelCG6829, Chronic Airflow Obstructions, postnatal development, classic hairy cell leukaemia, Nl1, CG7788, Monohydrate, crice, Guanidine Sulfate (2:1), Essigsaeure, protein, Hydrogenchlorid, Xkl-1, Cahp, lysis, peptide, Gsfsco1, B37, ClvPrd, peptido, Ccne, Glacial Acetic Acid, chronic obstructive airways disease NOS (disorder), NL1, Min, Apaf1, NL2, Chronic Obstructive Pulmonary Disease (COPD), protein aggregate, Gsfsco5, ARK, DmelCG42273, SOW3, Peptone, Eubacteria, COLD (chronic obstructive lung disease), peptides, NEC in ICD9CM_2006, Rubidium, vps27, Acetic Acid, min, arc, SFRS5, mAPC, Phosphate, 4' DDT, ark, T1, Trichloroacetic, Chronic airflow limitation, 2-trichloroethylidene)bis(4-chloro)-, ICE, GLYCINE RICH PROTEIN 7, papilla, l(2)k08110, trifluoro-, Sodium Trichloroacetate, ethoic acid, Strains, necrosis, Guanidine Sulfate (1:1), Lyw-57, dApaf1, glycine-rich RNA-binding protein 8, Sl, CHRONIC OBSTRUCTIVE PULMONARY DISEASE, CHRONIC, obstructive lung disease, Monohydrochloride, ice, iCE, D-Apaf-1, drice, lamina, Dmel_CG7826, flanges, Tr-kit, classic hairy cell leukemia, br37, beta-D-fructofuranosyl, Airflow Obstructions, pulmonary disease (COPD), NOD, SLP65, Ly-57, weight, MMEL2, drIce, drICE, low temperature, Xanthomonas campestris pv. oryza, CHRONIC OBSTRUCTIVE, 3.1.3.48, chronic obstructive pulmonary disease and allied conditions, HCL, kl1-A, Chronic airway disease, bacteriocin activity, NOS, p24, cultivar, KIT, CG2903, Dmel_CG7835, Acetic acid, COPD NOS, Mnb, MNB, CHRONIC OBSTRUCTIVE LUNG DIS, tyrosine-protein kinase Kit, Chronic airway obstruction, SLP-65, table sugar, growth pattern, BASH, cold, 1-alpha-D-Glucopyranosyl-2-beta-D-fructofuranoside, Phaseolus max, Step, non-developmental growth, shelves, HCl, P45, Trifluoroacetic, Guanidine Monohydrochloride, kit, hac1, AW124434, Sid470p, dapaf-1S, cv. Wye, Chronic Airflow Obstruction, apaf-1, 1'-(2, sensory visual perception, lysin activity, dapaf-1L, l35Dd, Cesium, spine, STEP, Acetamide, COPD - Chronic obstructive pulmonary disease, chronic obstructive airways disease, p45, Eph2, Polypeptide, obstructive pulmonary disease (COPD), AI046351, Strains and Sprains, COLD, chronic obstructive lung disease, E-260, Dapaf-1/HAC-1, DISEASE (COPD), SCF receptor activity, Gene, CH3-COOH, PLEXIN-B1, cycE, eubacteria, circadian rhythm, krk1., vac, l(2)br37, CYCLE, protrusion, Buffer, Dark/Hac-1/dApaf1, cytohesin/GRP1, Cold, Methanecarboxylic acid, Hac1, Dark/Hac-1/dApaf-1, Pulmonary Disease, polypeptide chain, scfr, Sacharose, Hgr, Sprains, SCFR, cloruro de hidrogeno, CH3CO2H, Pro-Mega, Guanidium Chloride, study, D12S755E, Fdc, Cold Temperatures, CG2916, CYCE, Saccharose, DmelCG3938, CyclE, dapaf-1, 3938, l(2)SH2 0323, dapaf, ridges, dark, Chronic Obstructive, Monera, l(2)k05007, dm-cycE, Neural-specific protein-tyrosine phosphatase, DARK, SELP, fungi, dArk, Dapaf-1, Guanidine Hydrochloride, 2-iodo-, BLNK-S, COPD, DYRK1, COAD - Chronic obstructive airways disease, AA960066, 1500003O03Rik, 4'-Dichlorodiphenyltrichloroethane, 3H-labeled, protein complex, 4'-DDT, not elsewhere classified, Sprain, l(2)23Ad, apaf1, Peptide, Guanidinium, PULM DIS CHRONIC OBSTRUCTIVE, Chronic obstructive pulmonary disease finding (finding), strain, development, sep5, [HCl], HOAc, natural protein, sucrose, Protein, Hydrochloride, Trichloroacetate, Guanidine Sulfite (1:1), Dyrk1, Rubidium Trichloroacetate, CyeE, CES2A1, Dark, GLYCINE-RICH RNA-BINDING PROTEIN 7, l(2)SH0323, Dark/Apaf-I, ACETONITRILE, flange, cyanomethane, CYH1, chronic obstructive airway disease, Temperature, c-KIT, E 260, Dops, CC1, l(2)35Dd, DmelCG2916, Tripcellim, acide acetique, beta-D-fructofuranosyl alpha-D-glucopyranoside, dApaf-1/DARK/HAC-1, Chlorophenothane, Interleukin-1 beta convertase, Protein Gene Products, process, Trypure, c-kit, p'-DDT, Cane sugar, concentration, velocity, 3.4.22.36, processus, Peptid, cold (chronic obstructive lung disease), HRS-2, INS No. 260, Chronic Obstructive Lung Disease, acetic acid, growth, CG3938, White sugar, dAPAF-1, UltrasonicDmelCG6525, PSENL3, IMP1, T18E12_21, MSTP086, signal peptide peptidase, "Phytomonas" Bergey et al. 1923, H13, proteomic analysis, PSL3, SPPL1, IMPAS-1, CG6525, dJ324O17.1, T18E12.21, SPP, Spp, spp, IMPAS, Xanthomonas albilineans, FBgn0082831, ATSPP.sodium salt, SH2-Bb, ammonium formate, 13C-labeled, cadmium salt, MeCN, ion, NCMe, selection process, instrument configuration, zinc formate, cleavage, Irip, lead salt, magnesium formate, protein, clefted, zinc salt, NAALAdase, Solvent, ATSPP., peptide, Polypeptides, NAALADase I, protein polypeptide chains, L-Isomer Methionine, FOLH, ammonium tetraformate, Methionine, IMP1, ClvPrd, peptido, BANF, cobalt(II) formate dihydrate, CH3-C#N, T18E12.21, C530001K22Rik, Min, Software Engineering, 3.4.17.21, protein aggregate, WMS, Computer Program, DmelCG42273, SH2-B, average, somitogenic mesoderm, Divorced, F, C79325, T18E12_21, peptides, Membrane glutamate carboxypeptidase, M, 2610036I19Rik, Open, 2610510L13Rik, min, Computer Programs and Programming, mAPC, Divorces, proteins, present in organism, NAALAD1, copper, SCAN, free, ecotype, Folylpoly-gamma-glutamate carboxypeptidase, AI047805, fSAP152, lithium salt, column, 380, H13, ACN, ppm, sample, Acn, n, blood capillary, GPHYSD2, z, Racemethionine, ammonium (2:1) salt, nickel salt, SGS, ratio, visualization, subdivided, cobalt (+2) salt, unsegmented mesenchyme, T5E21.12, PSL3, SPPL1, IMPAS-1, 2-Amino-4-(methylthio)butyric acid, Dmel_CG7826, Th, Normalcy, SH2-B PH domain-containing signaling mediator 1, fragmented, not genetically inherited, Source Softwares, Software Tools, ACMICD, Programs, Program, Computer Applications, sodium (4:1:1) salt, count, Ionen, Dm1, magnesium salt, PSM, Psm, Algorithm, Computer Applications Software, Computer Applications Softwares, Methionin, cultivar, methionine, Hmet, Softwares, presumptive somite mesoderm, Prostate-specific membrane antigen, nickel (+2) salt, fixed, Dmel_CG7835, Hydrogen Oxide, forked, T5E21_12, Mnb, MNB, parent ion, ammonium salt, Pteroylpoly-gamma-glutamate carboxypeptidase, Software Applications, divided, instrument, Source Software, FGCP, cracked, formate, proportionality, cobaltous formate, cromium (+3), rate, MASS, AW124434, septate, beta Trypsin, Pro-rich, nanospray, data encoding as image, Applications, Health, precursor ion, MSTP086, copper salt, ammonium (4:1) salt, PSMA, Polypeptide, methyl cyanide, IMPAS, lead (+2) salt, Liquimeth, time, Proteomes, Computer Software Applications, SH2 domain-containing protein 1B, 2-amino-4-(methylsulfanyl)butanoic acid, nickel formate dihydrate, lead formate, False, Peptidomics, fractured, aluminum salt, cesium salt, unsegmented paraxial mesoderm, L Isomer, FBN, Gene, ACINUS, precursor, Computer, thomson, protein-containing complex, somitomeric mesoderm, GCPII, CG7826, formic acid, mass-to-charge ratio, period, potassium salt, polypeptide chain, ECTOL1, 14C-labeled, CG7835, Gene Products, CG42273, L-Methionine, N-acetylated-alpha-linked acidic dipeptidase I, mKIAA1299, Separated, Pedameth, Application, Normalities, Open Source Softwares, SH2B, proportion, DmelCG6525, Acinus, thallium (+1) salt, iones, Software Application, rubidium salt, beta-Trypsin, Open Source Software, mGCP, calcium formate, methanoic acid, ions, FBgn0082831, Probabilities, Computer Software Application, acinusL, OCTD, PSENL3, 2-amino-4-(methylthio)butanoic acid, 10^[-6], Tools, acinusS, dJ324O17.1, peptidos, cromium (+3) salt, metionina, sodium formate, Applications Software, Open Source, DYRK1, data, Computer Software, nickel formate, 1-(14)C-labeled, 3H-labeled, signal peptide peptidase, DL-Methionine, protein complex, Proteins, SPP, Spp, split, strontium formate, total expressed protein, potassium formate, copper (+2) salt, backward, strontium salt, acetonitrile, L-Isomer, Peptide, mKIAA0670, Tool, GCP2, strain, Glutamate carboxypeptidase II, polypeptide, Software Tool, Cell growth-inhibiting gene 27 protein, capillary vessel, Temperatures, native protein, natural protein, cupric formate, Protein, hemorrhaged, spp, MFS1, Dyrk1, segmental plate, aluminum formate, Sh2bpsm1, Software, ACETONITRILE, cyanomethane, WMS2, Data Base, Met, torn, AU020952, Separation, 10^[-9], PH and SH2 domain-containing signaling mediator, lithium formate, Normality, Separations, CC1, Engineering, Tripcellim, ethanenitrile, sample population, chromic formate, Folate hydrolase 1, ME-IV, Protein Gene Products, Computer Programs, Trypure, Gene Proteins, Applications Softwares, Ion, SSKS, AI425885, calcium salt, quotient, alpha-amino-gamma-methylmercaptobutyric acid, CG6525, Peptid, SCH, reversedtotal expressed protein, species, "Pseudomonas oryzae" Uyeda and Ishiyama in Ishiyama 1926., Xanthomonas albilineans, Proteomes, "Phytomonas" Bergey et al. 1923Data Set., nutrient, bacteria, data, taxonomy, Peptidomics, protein complex, exits through, Proteins, Systematics, Gene, eubacteria, protein, protein-containing complex, infectivity, results, Classifications, Taxonomies, Readability, protein polypeptide chains, hierarchies, hierarchy, native protein, natural protein, polypeptide chain, systematics, Xanthomonas campestris pv. oryza, Protein, proteomic analysis, Gene Products, Pathogenicity, prokaryotes, protein aggregate, viral infection, Taxonomy, Eubacteria, Gene Expressions, nutrients, proteins, virus process, Understanding, exposed, Expressions, Monera, free, Protein Gene Products, Gene Proteins, extruding from, Bacteria <bacteria>, Prokaryotae, prokaryote, "Phytomonas" Bergey et al. 1923, virulence, fungi, Procaryotae, Expression, species, Xanthomonas albilineans, Prokaryotatotal expressed protein, assay, Xanthomonas albilineans, determination, Proteomes, "Phytomonas" Bergey et al. 1923, species., chemical analysis317trueComparative proteome analysis in different media using three Xanthomonas speciesPurpose -Comparison of proteome incubated in different media (rich and minimal media) -Three Xanthomonas species were used (Xanthomonas oryzae pv. oryaze, X. campestris pv. vesicatoria, and X. axonopodis pv. glycines. -Shotgun proteomic was 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