Dataset Information

Characterization of oligopeptides in Solanum lycopersicum xylem exudates

ABSTRACT: We collected 120 ml of xylem exudate from the cut surface of the first internodes from the hypocotyls of the tomato plants at 45 DAG for five hours. The exudates were purified using the gel-free method that we developed previously (Okamoto et al., 2015, Plant J.). Using gel filtration chromatography (Superdex Peptide 10/300 GL gel filtration column (10 mm  300 mm; GE Healthcare)), we separated small-protein-containing fractions (small-protein fractions) and oligopeptide-containing fractions (peptide fractions). The small-protein fractions were treated with trypsin, and the peptide fractions were further purified with multimode chromatography (Asahipak GS-320 column (7.5 mm  300 mm; Shodex)). Then, data-dependent MS/MS (ddMS/MS) was performed using an EASY-nLC 1000 (Thermo Fisher Scientific) connected to an Orbitrap Elite mass spectrometer (Thermo Fisher Scientific).

ORGANISM(S): Cellular Organisms

SUBMITTER: Satoru Okamoto

PROVIDER: PXD032703 | JPOST Repository | Fri Apr 15 00:00:00 BST 2022

REPOSITORIES: jPOST

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			Action	DRS
	181112_okamoto_Slycoper_10_2C67_10.raw	Raw
	181112_okamoto_Slycoper_10_2C67_11.raw	Raw
	181112_okamoto_Slycoper_10_2C67_12.raw	Raw
	181112_okamoto_Slycoper_10_2C67_13.raw	Raw
	181112_okamoto_Slycoper_10_2C67_14.raw	Raw

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Project description:Flooding is a serious problem for soybean cultivation because it markedly reduces growth. We found that treatment with ABA enhances survival ratio of soybean under flooding stress. To investigate the role of ABA in soybean under flooding stress, proteomic changes were analyzed. Total protein and nuclear protein fractions from root tips of soybean seedlings that treated with flooding or flooding with ABA were analyzed. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin and lysyl endopeptidase. Resulting peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Using an Ultimate 3,000 nanoLC system (Dionex), peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and their separation and spraying were done using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3, Nikkyo Technos) with a spray voltage of 1.5 kV. The elution was done with a linear acetonitrile gradient 8-30% in 120 min for gel free proteomics in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over a mass range of 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy. The top 10 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 sec to prevent repetitive selection of peptides.Identification of proteins were performed by Mascot search engine (version 2.3.0.2, Matrix Science) using soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0)and a common contaminants database (262 sequences). Parameters used in Mascot searches were follows: Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charge was set at +2, +3, and +4. Automatic decoy database search was performed in the search. Mascot results were filtered with Mascot percolator to improve accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were less than 1.0%. The Mascot results were used for differential analysis using SIEVE software (version 2.0, Thermo Fisher Scientific).

Project description:Cells were treated with DMSO (biological triplicate) or degrader at indicated dose and time (Meta Treatment Table) and cells were harvested by centrifugation. Lysis buffer (8 M Urea, 50 mM NaCl, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (EPPS) pH 8.5, Protease and Phosphatase inhibitors from Roche) was added to the cell pellets and homogenized by 20 passes through a 21 gauge (1.25 in. long) needle to achieve a cell lysate with a protein concentration between 1 - 4 mg/mL. A micro-BCA assay (Pierce) was used to determine the final protein concentration in the cell lysate. 200 µg of protein for each sample were reduced and alkylated as previously described1. Proteins were precipitated using methanol/chloroform. In brief, four volumes of methanol were added to the cell lysate, followed by one volume of chloroform, and finally three volumes of water. The mixture was vortexed and centrifuged to separate the chloroform phase from the aqueous phase. The precipitated protein was washed with three volumes of methanol, centrifuged and the resulting washed precipitated protein was allowed to air dry. Precipitated protein was resuspended in 4 M Urea, 50 mM HEPES pH 7.4, followed by dilution to 1 M urea with the addition of 200 mM EPPS, pH 8. Proteins were first digested with LysC (1:50; enzyme:protein) for 12 hours at room temperature. The LysC digestion was diluted to 0.5 M Urea with 200 mM EPPS pH 8 followed by digestion with trypsin (1:50; enzyme:protein) for 6 hours at 37 °C. Tandem mass tag (TMT) reagents (Thermo Fisher Scientific) were dissolved in anhydrous acetonitrile (ACN) according to manufacturer’s instructions. Anhydrous ACN was added to each peptide sample to a final concentration of 30% v/v, and labeling was induced with the addition of TMT reagent to each sample at a ratio of 1:4 peptide:TMT label. The 11 or 16-plex labeling reactions were performed for 1.5 hours at room temperature and the reaction quenched by the addition of hydroxylamine to a final concentration of 0.3% for 15 minutes at room temperature. Each of the sample channels were combined in a 1:1 ratio, desalted using C18 solid phase extraction cartridges (Waters) and analyzed by LC-MS for channel ratio comparison. Samples were then combined using the adjusted volumes determined in the channel ratio analysis and dried down in a speed vacuum. The combined sample was then resuspended in 1% formic acid and acidified (pH 2 - 3) before being subjected to desalting with C18 SPE (Sep-Pak, Waters). Samples were then offline fractionated into 96 fractions by high pH reverse-phase HPLC (Agilent LC1260) through an aeris peptide xb-c18 column (phenomenex) with mobile phase A containing 5% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O, and mobile phase B containing 90% acetonitrile and 10 mM NH4HCO3 in LC-MS grade H2O (both pH 8.0). The 96 resulting fractions were then pooled in a non-continuous manner into 24 fractions and these fractions were used for subsequent mass spectrometry analysis. Data were collected using an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with a Proxeon EASY-nLC 1200 LC pump (Thermo Fisher Scientific) or an Orbitrap Eclipse Tribrid mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA) coupled with an UltiMate 3000 RSLCnano System. Peptides were separated on an EasySpray ES803a/ES803a.rev2 75 μm inner diameter microcapillary column (Thermo Fisher Scientific) or a 100 µm inner diameter microcapillary column packed with ~ 50 cm of Accucore C18 resin (2.6 µM, 100 Å, Thermo Fisher Scientific). Peptides were separated using a 190 min gradient of 6 - 27% acetonitrile in 1.0% formic acid with a flow rate of 300 or 350 nL/min. Each analysis used an MS3-based TMT method as described previously. The data were acquired using a mass range of m/z 340 – 1350, resolution 120,000, AGC target 5 x 105, maximum injection time 100 ms, dynamic exclusion of 120 seconds for the peptide measurements in the Orbitrap. Data dependent MS2 spectra were acquired in the ion trap with a normalized collision energy (NCE) set at 35%, AGC target set to 1.8 x 104 and a maximum injection time of 120 ms. MS3 scans were acquired in the Orbitrap with a HCD collision energy set to 55%, AGC target set to 2 x 105, maximum injection time of 150 ms, resolution at 50,000 and with a maximum synchronous precursor selection (SPS) precursors set to 10.

Project description:Flooding injury is one of the abiotic constraints on soybean growth. An experimental system established for evaluating flooding injury in soybean seedlings indicated that the degree of injury is dependent on seedling density in floodwater. To understand the molecular mechanism responsible for the injury, proteomic alterations in soybean seedlings that correlated with severity of stress were analyzed using label-free quantitative proteomics. Two-day-old soybeans, seedlings flooded for 2 days and hypocotyls of seedlings grown for 3 days after flooding were analyzed. The radicles of soybean seedlings were separated to root tips (RT), roots without tip (RwoT) and hypocotyls (Hypo). Protein was extracted from these organs by TCA/Acetone method. Protein samples were cleaned up with chloroform/methanol method and then subjected to in-solution digestion with trypsin. Resulting peptides were analyzed on a nanospray LTQ XL Orbitrap MS (Thermo Fisher Scientific) operated in data-dependent acquisition mode with the installed Xcalibur software (version 2.0.7, Thermo Fisher Scientific). Using an Ultimate 3,000 nanoLC system (Dionex), peptides in 0.1% formic acid were loaded onto a C18 PepMap trap column (300 µm ID × 5 mm, Dionex). The peptides were eluted from the trap column and their separation and spraying were done using 0.1% formic acid in acetonitrile at a flow rate of 200 nL/min on a C18 Tip column (75 µm 1D × 120 mm, nano HPLC capillary column, NTTC-360/75-3, Nikkyo Technos) with a spray voltage of 1.5 kV. The elution was done with a linear acetonitrile gradient 8-30% in 120 min for gel free proteomics in 0.1% formic acid. Full-scan mass spectra were acquired in the Orbitrap over a mass range of 400-1,500 m/z with a resolution of 30,000. A lock mass function was used to obtain high mass accuracy. The top 6 most intense precursor ions were selected for collision-induced fragmentation in the linear ion trap at normalized collision energy of 35%. Dynamic exclusion was employed within 90 sec to prevent repetitive selection of peptides.Identification of proteins were performed by Mascot search engine (version 2.3.0.2, Matrix Science) using soybean peptide database (55,787 sequences) obtained from the soybean genome database (Phytozome version 8.0)and a common contaminants database (262 sequences). Parameters used in Mascot searches were follows: Carbamidomethylation of cysteine was set as a fixed modification, and oxidation of methionine was set as a variable modification. Trypsin was specified as the proteolytic enzyme and one missed cleavage was allowed. Peptide mass tolerance was set at 5 ppm, fragment mass tolerance was set at 0.5 Da, and peptide charge was set at +2, +3, and +4. Automatic decoy database search was performed in the search. Mascot results were filtered with Mascot percolator to improve accuracy and sensitivity in the peptide identification. False discovery rates for peptide identification of all searches were less than 1.0%. The Mascot results were used for differential analysis using SIEVE software (version 1.3, Thermo Fisher Scientific)

Project description:2-cell (E1.5) stage embryos were cultured in normal KSOM+AA conditions until E3.5 +2h and thereafter 100 embryos each were moved to control or p38-MAPKi conditions and cultured for another 7 hours (E3.5 +9h). The embryos were then washed through pre-warmed (37˚C) Hank’s balanced salt solution (HBSS, Sigma-Aldrich; Cat. No. H9269) and lysed by moving to a 1.5ml centrifuge tube containing about 15µl of SDT-lysis buffer (4% (w/v) SDS, 100 mM Tris-HCl pH 7.6, 0.1 M DTT). Cell lysis was performed by incubating the tubes in a 95˚C thermoblock for 12 minutes, brief centrifugation at 750 rpm, cooling to room temperature and storage at -80˚C. Individual protein solutions were processed by filter-aided sample preparation (FASP) method with some modifications (see the associated publication for more details). Resulting peptides were cleaned by liquid-liquid extraction (3 iterations) using water saturated ethyl acetate. 1/10th of the FASP eluate was taken out for direct LC-MS measurements, evaporated completely in SpeedVac concentrator (Thermo Fisher Scientific). Peptides were further transferred into LC-MS vials using 50μL of 2.5% formic acid (FA) in 50% acetonitrile (ACN) and 100μL of pure ACN and with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator. Phosphopeptides were enriched from the remaining 9/10th of the cleaned FASP eluate after complete solvent evaporation (SpeedVac concentrator) using High-Select™ TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific) according to manufacturer protocol. Phosphopeptide standards (0.1 pmol of MS PhosphoMix 1, 2, 3 Light; Sigma-Aldrich, St. Louis, Missouri, USA) was added to suspended sample in binding/equilibration buffer. Flow-through fraction was dried and used for second enrichment step using High-Select™ Fe-NTA Phosphopeptide Enrichment Kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to manufacturer protocol. Resulting phosphopeptides were extracted into LC-MS vials by 2.5% FA in 50% ACN and 100% ACN with addition of polyethylene glycol (final concentration 0.001%) and concentrated in a SpeedVac concentrator). LC-MS/MS analyses of all peptide mixtures (2 peptide solutions for each sample – 1) not enriched; 2) enriched on phosphopeptides using TiO2 enrichment kit) were done using RSLCnano system connected to Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific). Prior to LC separation, tryptic digests were online concentrated and desalted using trapping column (100 μm × 30 mm, column compartment temperature of 40˚C) filled with 3.5-μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow rate - 300nl/min) from the trapping column onto an analytical column (Acclaim Pepmap100 C18, 3 µm particles, 75 μm × 500 mm; column compartment temperature of 40˚C, Thermo Fisher Scientific) by using 50 or 100 minutes long nonlinear gradient program (1-56% of mobile phase B; mobile phase A: 0.1% FA in water; mobile phase B: 0.1% FA in 80% ACN) for analysis of phosphopeptide enriched fractions or not enriched peptide mixtures, respectively. Equilibration of the trapping column and the analytical column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 (New Objective) ion source with sheath gas option and SilicaTip emitter (New Objective; FS360-20-15-N-20-C12) utilization. ABIRD (ESI Source Solutions) was installed. MS data were acquired in a data-dependent strategy with cycle time for 3 seconds and with survey scan (350-2000 m/z). The resolution of the survey scan was 60000 (200 m/z) with a target value of 4×105 ions and maximum injection time of 50ms. HCD MS/MS (30% relative fragmentation energy, normal mass range) spectra were acquired with a target value of 5.0x104. The MS/MS spectra were recorded in Orbitrap at resolving power of 30,000 or 15,000 (200 m/z) and the maximum injection time for MS/MS was 500 or 22 ms for analysis of phosphopeptides enriched fraction or non-enriched peptide mixture, respectively. Dynamic exclusion was enabled for 60 seconds after one MS/MS spectra acquisition. The isolation window for MS/MS fragmentation was set to 1.6 m/z.

Dataset Information

Characterization of oligopeptides in Solanum lycopersicum xylem exudates

Dataset's files

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