Proteinscape v3

The MS/MS data, formatted as Mascot Generic Format (mgf), was imported into ProteinScape™ V3.1 (Bruker Daltonics, Billerica, MA, USA) proteomics data analysis software for downstream mining of the NCBIprot database, using ‘Eukaryotes’ as the taxonomical search parameter. Database searches were conducted utilising the Mascot™ V2.5.1 (Matrix Science, Boston, MA, USA) search engine. Mascot search parameters were set in accordance with published guidelines [10 (link)] and to this end, fixed (carbamidomethyl “C”) and variable (oxidation “M” and deamidation “N, Q”) modifications were selected along with peptide (MS) and secondary fragmentation (MS/MS) mass tolerance values of 0.5 Da whilst allowing for a single 13C isotope. Using these criteria Mascot deemed scores over 68 to be significant. From the protein lists produced by Mascot the individual protein identifications scoring over 68 were inspected manually and considered significant only if (i) two peptides were matched for each protein; (ii) peptides were represented by a sequence coverage of > 5%; and (iii) each matched peptide contained an unbroken “b” or “y” ion series represented by a minimum of four contiguous amino acid residues.

Deconvoluted MS/MS data in Mascot Generic Format (.mgf) were imported into ProteinScape^™ V3.1 (Bruker UK) proteomics data analysis software for downstream database mining of a cognate C. abortus genomic sequence utilising the Mascot^™ V2.3 (Matrix Science) search algorithm. The protein content of each individual gel slice was established using the “Protein Search” feature of ProteinScape^™, whilst separate compilations of the proteins contained in all 26 gel slices for each sample were produced using the “Protein Extractor” feature of the software. Mascot search parameters were set in accordance with published guidelines [26 (link)] and to this end, fixed (carbamidomethyl “C”) and variable (oxidation “M” and deamidation “N,Q”) modifications were selected along with peptide (MS) and secondary fragmentation (MS/MS) tolerance values of 0.5 Da whilst allowing for a single 13C isotope. Molecular weight search (MOWSE) scores attained for individual protein identifications were inspected manually and considered significant only if: (a) two peptides were matched for each protein; and (b) each matched peptide contained an unbroken “b” or “y” ion series represented by of a minimum of four contiguous amino acid residues.

Deconvoluted MS/MS data in .mgf (Mascot Generic Format) format were imported into ProteinScape™ V3.1 (Bruker Daltonics) proteomics data analysis software for downstream mining of a custom L. salmonis database. This custom database was constructed from all the “Lepeophtheirus salmonis” protein entries found in the NCBInr database as of January 2018 and comprised 38,092 sequences in total. Database searches were conducted utilising the Mascot™ V2.54.1 (Matrix Science) search engine. Mascot search parameters were set in accordance with published guidelines [25 (link)] and to this end, fixed (carbamidomethyl “C”) and variable (oxidation “M” and deamidation “N,Q”) modifications were selected along with peptide (MS) and secondary fragmentation (MS/MS) tolerance values of 0.5 Da whilst allowing for a single 13C isotope. Protein identifications obtained from each of the 25 individual gel slices per lane were compiled using the “protein list compilation” feature within Proteinscape [26 (link)]. From the compiled protein lists individual protein identifications were inspected manually and considered significant only if: (i) two peptides were matched for each protein; (ii) peptides were represented by a sequence coverage of > 5%; and (iii) each matched peptide contained an unbroken “b” or “y” ion series represented by a minimum of four contiguous amino acid residues.