Computational protocol: Quantitative Proteomics Study of Larval Settlement in the Barnacle Balanus amphitrite

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Protocol publication

[…] The raw MS data were converted into mascot generic files using Proteome Discover (1.2) and then submitted to Mascot version 2.2 (Matrix Sciences Ltd., London, UK) for searching against an in-house protein database developed in our transcriptome study . The mass tolerances were set at 10 ppm for the peptide precursors and 0.5 Da for the fragment ions. A decoy option was included. Carboxamidomethylation at cysteine residues was set as a fixed modification, and oxidation at methionine residues was set as a variable modification. Up to 1 missed trypsin cleavage was permitted.The resulting .dat files from the Mascot search were processed using Scaffold (version 4.0, Proteome Software Inc., OR, USA) to validate the MS/MS identification. Peptide identification was accepted if the result could established at >95.0% probability by the Scaffold Local FDR, whereas protein identification was accepted if the result had a probability of >99.0% and contained at least 1 identified peptide. Peptide and protein probabilities assigned by the PeptideProphet and ProteinProphet . Protein XML files were then exported from Scaffold to calculate the protein abundances were calculated using APEX quantitation proteomics tools , . A 1.5-fold change was set as the cutoff, and only proteins with >5,000 molecules per cell during at least one stage were considered for significantly up-regulated proteins and proteins with >5,000 molecules per cell during at least 3 stages were considered for significantly down-regulated proteins . [...] Amino acid sequences of vitellogenin from various species were downloaded from GenBank and aligned using MUSCLE via the CIPRES Portal v2.2 . Neighbor-joining analysis was performed with MEGA4. All of the positions containing alignment gaps and missing data were eliminated in pairwise sequence comparisons (pairwise deletion option). The topological stability was evaluated based on 1,000 bootstrapping (BS) replications. […]

Pipeline specifications

Software tools MUSCLE, CIPRES Science Gateway, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Drosophila melanogaster