Computational protocol: Isolation and identification of ubiquitin-related proteins from Arabidopsis seedlings

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Protocol publication

[…] The eluted proteins from three independent purifications were mixed and fractionated by SDS-PAGE. Protein bands were detected with Flamingo™ Fluorescent Gel Stain (Bio-Rad Laboratories, CA, USA). The protein bands were excised and other smearing regions were cut into 2-mm-long gel pieces for in-gel trypsin digestion. In-gel digestion and MS/MS analysis were performed according to the methods described by and . The gel pieces were dehydrated by washing twice with 100% acetonitrile, and dried with a vacuum concentrator. The proteins were reduced with 10 mM DTT at 56 °C for 45 min and then alkylated with 55 mM iodoacetamide at room temperature in the dark for 30 min. After washing twice with 25 mM ammonium bicarbonate, the samples were dehydrated again with 50% acetonitrile and dried. The protein samples were digested with 10 μg ml−1 proteomics-grade trypsin (Promega, Madison, WI, USA) for 12 h at 37 °C.The digested peptides were subjected to column chromatography (PEPMAPC18, 5 μm, 75 μm internal diameter, 15 cm; Dionex, Sunnyvale, CA) using the CapLC system (Waters, Milford, MA, USA). Buffers were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A linear gradient from 5% to 45% B for 25 min was applied, and peptides eluted from the column were introduced directly into a Q-TOF Ultima mass spectrometer (Waters) at a flow rate of 100 nl min−1. In the ESI-positive ion mode, ionization was performed at a capillary voltage of 2.2 kV with the PicoTip nanospray source (New Objective, Cambridge, MA). For survey scan, mass spectra were acquired for the two most intense ions from the precursor ion scan between m/z 400 and 1500. For collision-induced dissociation (CID), the collision energy was set automatically according to the mass and charge state of the precursor peptide. MS/MS spectra were analysed with the MASCOT server against a protein database from the National Center for Biotechnology Information. The applied MASCOT search parameters were as follows: (i) taxonomy: Arabidopsis thaliana; (ii) potential modifications: carbamidomethyl and oxidation as fixed modifications, myristoylation (N-term G, K); (iii) max missed cleavage: 1; (iv) peptide tolerance: ±0.5 Da; (v) MS/MS tolerance: ±0.2 Da; and (vi) peptide charge: 2+ and 3+. Proteins detected from peptide fragments with high reliability [MASCOT score >40 (P <0.05)] were selected as identified proteins.The presence of putative motifs in the identified proteins was analysed using Eukaryotic Linear Motif resource (ELM; http://elm.eu.org/) for destruction-box (D-box) and KEN-box, and GENETYX-MAC software for PEST sequences. […]

Pipeline specifications

Software tools Mascot Server, ELM
Application MS-based untargeted proteomics
Organisms Arabidopsis thaliana