Computational protocol: In-depth characterization of trypsin-like serine peptidases in the midgut of the sugar fed Culex quinquefasciatus

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Protocol publication

[…] Mass spectra were searched against a customized non-redundant database including sequences of all Culicidae species available at UniRef100 (101,993 sequences, downloaded May 2015, using the Mascot MS/MS ion search engine (Matrix Science, Oxford, UK, version 2.4.1). The search parameters in the Mascot server were as follows: lack of taxonomic restrictions; one tryptic missed cleavage; carbamidomethylation of cysteine residues as a fixed modification and oxidation of methionine and acetylation as variable modifications; 10 ppm mass tolerance for the MS mode and 0.5 Da tolerance for its corresponding MS/MS fragments. Scaffold (version 4.3.0, Proteome Software Inc., Portland) was used to validate MS/MS peptide and protein identifications. Peptide identifications were accepted at 95.0 % probability by the Peptide Prophet algorithm [] using the Scaffold delta mass correction. Protein identifications were accepted at 95.0 % probability and if they were supported by two or more independent pieces of evidence (e.g., identification of a peptide with different charge states, a modified and a non-modified version of the same peptide, or two different peptides). Protein probabilities were assigned by the Protein Prophet algorithm [].To confirm peptidase identifications, mass spectra were also analyzed using the ProLuCID 1.3 engine at the PatternLab platform [] against the same customized database. Searches were performed with one missed cleavage, with carbamidomethylation of cysteine residues as a fixed modification, methionine oxidation as a variable modification and mass tolerances of 40 ppm and 0.5 Da for precursor and fragment ions, respectively. The validity of the peptide sequence matches (PSMs) was assessed using the Search Engine Processor (SEPro) at the PatternLab platform []. […]

Pipeline specifications

Software tools Mascot Server, ProLuCID, PatternLab, SEPro
Application MS-based untargeted proteomics
Organisms Culex quinquefasciatus, Homo sapiens, Canis lupus familiaris