Computational protocol: De novo subtype and strain identification of botulinum neurotoxin type B through toxin proteomics

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Protocol publication

[…] Peak lists for all MS/MS spectra were extracted using Mascot Distiller (Matrix Science Inc., Boston, MA, USA, version 2.2.0) and merged together using Mascot Daemon (Matrix Science Inc, Boston, MA, USA, version 2.2.2). Database searches were conducted using Mascot (Matrix Science Inc, Boston, MA, USA, version 2.2.0) against a database generated by extracting entries from the NCBI non-redundant database using “Clostridia” as the extraction parameter. Mascot parameters were applied as follows for standard protein database searching: nine maximum missed cleavages, non-specific enzyme, peptides with up to +4 charge, MS1 tolerance of 20 ppm and MS/MS tolerance of 0.8 Da, and carbamidomethyl, deamidation, and oxidation modifications allowed. Mascot parameters were applied as follows for amino acid substitution database searching: nine maximum missed cleavages, semi-tryptic (K and R) and semi-chymotryptic enzyme selectivity, peptides with up to +4 charge, MS1 tolerance of 20 ppm and MS/MS tolerance of 0.5 Da, semi-specific searches included one non-specific cleavage site located on either the N or C terminus of the peptide. MS/MS-based peptide and protein identifications were validated using Scaffold (version Scaffold_2_00_06, Proteome Software Inc., Portland, OR, USA). Peptide identifications were accepted if they could be established at greater than 95.0% probability as specified by the Peptide Prophet algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability as specified by the Protein Prophet algorithm, and if they contained at least three peptides with unique amino acid sequences. […]

Pipeline specifications

Software tools Mascot Distiller, Mascot Server
Application MS-based untargeted proteomics
Chemicals Amino Acids