Computational protocol: Elucidation of Cross-Talk and Specificity of Early Response Mechanisms to Salt and PEG-Simulated Drought Stresses in Brassica napus Using Comparative Proteomic Analysis

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Protocol publication

[…] The raw data files acquired from the Orbitrap were converted to MGF files using Proteome Discoverer 1.2 (Thermo Fisher Scientific, Bremen, Germany). Protein identification was performed using the Mascot search engine (version 2.3.02; Matrix Science, London, UK) against two B. napus genome databases of an unpublished database containing 83,006 sequences and a published database containing 98,762 sequences (http://www.genoscope.cns.fr/brassicanapus/data/). Mass tolerance of 0.05 Da (ppm) was permitted for intact peptide masses and 0.1 Da for fragmented ions, with one missed cleavage allowed in the trypsin digests. Gln->pyro-Glu (N-term Q), oxidation (M), and deamidated (NQ) were selected as the potential variable modifications, and carbamidomethyl (C), iTRAQ8plex (N-term), and iTRAQ8plex (K) were selected as fixed modifications. Specifically, an automatic decoy database search was performed in Mascot by choosing the decoy checkbox in which a random sequence from the database was generated and tested for the raw spectra and the true database. To reduce the probability of false peptide identification, only peptides with a significance score ≥20 at the 99% confidence interval from a Mascot probability analysis greater than “identity” were counted.The minimum requirements for differentially expressed proteins (DEPs) were at least two matched unique peptides and a significant change (P < 0.05 and > or < 1.2-fold) in protein quantities between the stress-treated samples and control samples in at least one replicate, with the other replicate displaying a similar trend. Functional annotation of DEPs was conducted using the Blast2GO program against NCBInr and Swiss-Prot databases. The Kyoto Encyclopedia of Genes, Genomes database (KEGG; http://www.genome.jp/kegg/) and the Clusters of Orthologous Groups of Proteins database (COG; http://www.ncbi.nlm.nih.gov/COG/) were used to classify the proteins. [...] MRM was used to validate the DEPs. MRM transitions for selected proteins of interest were predicted using Skyline based on a protein FASTA database derived from a MS/MS spectra library imported from ProteinPilot software (AB SCIEX, Foster City, CA, USA) []. Samples were digested as described elsewhere [] and spiked with 20 fmol of α-galactosidase for data normalization. MRM analyses were performed on a QTRAP 5500 mass spectrometer (AB Sciex Inc., Foster City, CA, USA) equipped with a Waters nanoACQUITY ultra-performance liquid chromatography system (Waters Corp., Milford, MA). The mobile phase consisted of 0.1% aqueous formic acid (solvent A) and 98% acetonitrile with 0.1% formic acid (solvent B). Peptides were separated on a BEH130 C18 column (0.075 × 200 mm column, 1.7 μm; Waters) at 300 nL/min, and eluted with a gradient of 5%−8% solvent B for 2 min, 8%−30% solvent B for 94 min, and 30%−80% solvent B for 3 min. For QTRAP 5500 mass spectrometry, a spray voltage of 2100 V, nebulizer gas of 20 p.s.i., and dwell time of 10 ms were applied. Multiple MRM transitions were monitored using a unit resolution in both Q1 and Q3 quadrupoles to maximize specificity. Data analysis was conducted using Skyline []. The three most abundant transitions for each peptide were used for quantitation unless interference from the matrix was observed. […]

Pipeline specifications

Software tools Proteome Discoverer, Mascot Server, Blast2GO, ProteinPilot
Databases COGs UniProt KEGG
Application MS-based untargeted proteomics
Organisms Brassica napus
Chemicals Calcium, Polyethylene Glycols, Sodium Chloride