Computational protocol: The Non JAZ TIFY Protein TIFY8 from Arabidopsis thaliana Is a Transcriptional Repressor

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[…] A downscaled purification protocol was used. In short, cell extracts were made on 2.5 g cell culture and cleared by two subsequent centrifugation steps at 36,900×g for 20 minutes. In the first purification step, a protein input of 25 mg was incubated with 25 µl of IgG-Sepharose 6 Fast Flow beads (GE Healthcare). For the second step, 25 µl of Streptavidin Sepharose High Performance (Amersham) was used. Final elution was done with 40 µl 1× NuPAGE sample buffer containing 20 mM Desthiobiotin for 5 minutes. Beads were separated from eluate in a 1-ml Mobicol column (MoBiTec, Göttingen, Germany).Eluted proteins were separated in a short run of 7 minutes on a 4–12% gradient NuPAGE gel (Invitrogen) and visualized with colloidal Coomassie Brilliant Blue staining. The protein gel was washed for 2 hours in H2O, polypeptide disulphide bridges were reduced for 40 min in 25 mL of 6,66 mM DTT in 50 mM NH4HCO3 and sequentially the thiol groups were alkylated for 30 min in 25 mL 55 mM IAM in 50 mM NH4HCO3. After washing with H2O, a broad zone containing the proteins was cut from the protein gel, sliced into 24 gel plugs, and collected together in a single Eppendorf. Gel plugs were washed twice with H2O, dehydrated with 95% CH3CN (v/v), rehydrated with H2O and dehydrated again with 95% CH3CN (v/v). Dehydrated gel particles were rehydrated in 60 µL digest buffer containing 750 ng trypsin (MS Gold; Promega, Madison, WI), 50 mM NH4HCO3 and 10% CH3CN (v/v) for 30 min at 4°C. Proteins were digested at 37°C for 3.5 hours.The obtained peptide mixtures were introduced into an LC-MS/MS system, the Ultimate 3000 RSLC nano (Dionex, Amsterdam, The Netherlands) in-line connected to an LTQ Orbitrap Velos (Thermo Fisher Scientific, Bremen, Germany). The sample mixture was loaded on a trapping column (made in-house, 100 µm internal diameter (I.D.) ×20 mm (length), 5 µm C18 Reprosil-HD beads, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). After back-flushing from the trapping column, the sample was loaded on a reverse-phase column (made in-house, 75 µm I.D. ×150 mm, 5 µm C18 Reprosil-HD beads, Dr. Maisch). Peptides were loaded with solvent A (0.1% trifluoroacetic acid, 2% acetonitrile), and separated with a linear gradient from 2% solvent A' (0.1% formic acid) to 50% solvent B' (0.1% formic acid and 80% acetonitrile) at a flow rate of 300 nL/min, followed by a wash step reaching 100% solvent B'.The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS acquisition for the ten most abundant peaks in a given MS spectrum. In the LTQ Orbitrap Velos, full scan MS spectra were acquired in the Orbitrap at a target value of 1E6 with a resolution of 60,000. The ten most intense ions were then isolated for fragmentation in the linear ion trap, with a dynamic exclusion of 20 seconds. Peptides were fragmented after filling the ion trap at a target value of 1E4 ion counts.From the MS/MS data in each LC run, Mascot Generic Files were created using the Mascot Distiller software (version 2.4.1.0, Matrix Science, www.matrixscience.com/Distiller.html). When generating these peak lists, grouping of spectra was allowed with a maximum intermediate retention time of 30 seconds and a maximum intermediate scan count of 5 was used where possible. Grouping was done with 0.005 Da precursor tolerance. A peak list was only generated when the MS/MS spectrum contained more than 10 peaks. There was no de-isotoping and the relative signal-to-noise limit was set to 2. These peak lists were then searched with the Mascot search engine (version 2.3, MatrixScience, www.matrixscience.com) using the Mascot Daemon interface (Matrix Science, www.matrixscience.com). Spectra were searched against the TAIR10 database containing 35386 sequence entries. Variable modifications were set to methionine oxidation and methylation of aspartic acid and glutamic acid. Fixed modifications were set to carbamidomethylation of cysteines. Mass tolerance on MS was set to 10 ppm (with Mascot's C13 option set to 1) and the MS/MS tolerance at 0.5 Da. The peptide charge was set to 1+, 2+ and 3+ and the instrument setting was set to ESI-TRAP. Trypsin was set as the protease used, allowing for 1 missed cleavage, and also cleavage was allowed when arginine or lysine is followed by proline. Only high confident peptides, ranked one and with scores above the threshold score, set at 99% confidence, were withheld. Only proteins with at least two matched high confident peptides were retained.A list of non-specific background proteins was assembled by combining our previous background list with background proteins from control GS purifications on mock, GFP-GS, and GUS-GS cell culture extracts identified with LTQ Orbitrap Velos. To obtain the final list of interactors, these background proteins were subtracted from the list of identified proteins. […]

Pipeline specifications

Software tools Mascot Distiller, Mascot Server
Databases TAIR
Application MS-based untargeted proteomics
Organisms Pseudomonas syringae, Arabidopsis thaliana