Computational protocol: Mass Spectrometry Imaging and Identification of Peptides Associated with Cephalic Ganglia Regeneration in Schmidtea mediterranea*

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Protocol publication

[…] MS/MS base peak chromatograms from the HPLC-ESI-QTOF analysis were processed using DataAnalysis 4.2 (Bruker Daltonics) for compound spectra selection and charge deconvolution. The fragmentation spectra were exported as Mascot generic files (.mgf) and loaded into PEAKS Studio 7.0 (Bioinformatics Solutions Inc., Waterloo, ON, Canada) for searching against the Uniprot S. mediterranea protein database containing 712 entries, which includes all of the previously characterized neuropeptides (). The HPLC-ESI-FT-ICR MS data set was directly loaded into PEAKS Studio in the .raw format without preprocessing. The following search parameters were used, parent mass error tolerance, 50 ppm for spectra from QTOF and 15 ppm for spectra from FT-ICR MS; fragment mass error tolerance, 0.1 Da; charge options, no correction; enzyme, none; variable post-translational modifications (PTMs), acetylation (K), acetylation (N-term), acetylation (protein N-term), amidation, oxidation, pyro-Glu from Glu, pyro-Glu from Gln, and half of a disulfide bridge; maximum variable post-translational modifications per peptide, 3.Other than matches against the protein database, de novo sequence tags generated by PEAKS Studio were searched against the S. mediterranea transcriptome database () and BLAST searched against sequences from other species. Sequence tags were first filtered to keep only those with an average local confidence above 80%. Searching was further focused on matching residues having a local confidence higher than 80% to emphasize matching high quality portions of the sequence. For those without BLAST results, SignalP () was used to predict if a characteristic signal peptide was present in the sequence.In addition to using PEAKS Studio, database searching for the HPLC-ESI-FT-ICR MS data set was also conducted using ProsightPC 2.0 (Thermo Fisher Scientific) against the S. mediterranea Uniprot protein database (, ). Search parameters included an 82-Da precursor tolerance and 25 ppm fragment mass tolerance without protease specificity, and an E-value threshold of 1 × 10−4. […]

Pipeline specifications

Software tools PEAKS X, SignalP
Application MS-based untargeted proteomics
Organisms Schmidtea mediterranea
Diseases Multiple Sclerosis