Computational protocol: Exogenous Auxin Elicits Changes in the Arabidopsis thaliana Root Proteome in a Time-Dependent Manner

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Protocol publication

[…] Mass spectrometric data were processed and analyzed utilizing ProteinLynx Global Server v. 3.0.2 (PLGS, Waters Corporation). Chromatographic and mass spectrometric peak width resolutions were automatically determined using the software. Mass values were lock-mass corrected based on the exact m/z value of the +2 charge state of [Glu1]-fibrinopeptide B (785.842). Peaks were defined based on the low energy, elevated energy and bin intensity thresholds of 300, 20, and 1500 counts, respectively. Peak lists generated for each of the six SCX fractions from a sample were merged using PLGS after processing. The final peak list for each sample was then searched against a protein database containing the complete A. thaliana proteome including isoforms downloaded from UniProt (www.uniprot.org) and a randomized decoy entry for each real entry appended using PLGS. Workflow parameters for the protein identification searches were two possible missed cleavages utilizing trypsin as the protease, a fixed modification of carbamidomethylation of cysteine, possible modifications of carbamylation of lysine and the N-terminus of peptides, and oxidation of methionine. The software automatically determined peptide and peptide fragment mass tolerances and protein identifications were limited to less than 5% FDR. Information from the PLGS analysis was then processed and summarized using IsoQuant [] to resolve homology and quantitate proteins. Analysis in IsoQuant limited both peptide and protein identifications to less than 1% FDR. Protein quantitation using the Top3 method was limited to unique and razor peptides excluding those that resulted from in-source decay and allowed different peptides to be utilized for protein quantitation across samples. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [] partner repository with the dataset identifier PXD001400. […]

Pipeline specifications

Software tools PLGS, IsoQuant
Application MS-based untargeted proteomics
Organisms Arabidopsis thaliana