MassIVE MSV000100130

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Global Proteomics Analyses of Arogenate Dehydratase Knock-Out Mutants in Arabidopsis thaliana

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Description

Mass spectrometry-based proteomic datasets were generated from Arabidopsis thaliana Columbia (WT) plants and genetically defined T-DNA insertion knockout (KO) mutants obtained from SIGnAL. Plants were greenhouse-grown under controlled 16/8 h light/dark cycles with regular fertilization, and rosette leaves and stems were harvested at four weeks, flash-frozen, and stored at -80 deg C. Proteins were extracted from liquid-nitrogen-ground tissues using repeated methanolic ammonium acetate washes to remove metabolites, followed by solubilization in urea/thiourea/CHAPS buffer with TCEP. After sonication and clarification, protein concentrations were determined, samples were diluted into ammonium bicarbonate, and digested overnight with trypsin prior to chloroacetamide alkylation. Peptides were desalted using SCX and C18 solid-phase extraction and quantified before labeling. Peptides (30 ug per channel) were labeled with 8-plex iTRAQ reagents, combined, and fractionated offline using high-pH reversed-phase chromatography into concatenated fractions for LC-MS/MS analysis. Peptide separation employed a custom nanoflow LC system with dual trapping and analytical columns, coupled to an LTQ Orbitrap Velos mass spectrometer operated in data-dependent acquisition mode. Full MS scans were acquired at 30,000 resolution, followed by HCD MS/MS scans (7,500 resolution) of the top 10 precursors. Instrument data was processed using the FragPipe pipeline (MSFragger, MSBooster, Percolator, ProteinProphet, IonQuant, and TMT-Integrator) against the UniProt Arabidopsis database. Searches used semi-tryptic specificity, 20 ppm mass tolerances, iTRAQ-8 static modifications on Lys/N-termini, and standard variable modifications. Quantification was performed using TMT-Integrator with iTRAQ-8 settings, and results were filtered to 1% protein-level FDR. [doi:10.25345/C5QF8JZ35] [dataset license: CC0 1.0 Universal (CC0 1.0)]

Keywords: Arabidopsis ; Arogenate Dehydratase ; Stem ; Leaf ; DatasetType:Proteomics

Contact

Principal Investigators:
(in alphabetical order) 		James M. Fulcher, Pacific Northwest National Laboratory, USA
Submitting User: alchemistmatt
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