dependent collision-induced dissociation (CID) MS/MS scans
(1 μscans) using an isolation width of 3 amu and normalized
collision energy of 35%.
4. Fragmented masses are assigned during dynamic exclusion for
30 s after the second fragmentation event, and unassigned
charged ions are excluded from the MS/MS analysis.
3.4 MS Data
Processing
1. MS/MS spectra are analyzed using SEQUEST-HT™ (Proteome Discoverer 2.4 package, Thermo Fisher Scientific) against
the Teleostei UniProt/SwissProt protein database.
2. Use the following constraints for the searches: tryptic cleavage
with up to two missed cleavage sites and tolerances of 0.8 Da
for precursor ions and 0.6 Da for MS/MS fragment ions.
Carbamidomethylation (+57.021465 Da) on cysteine is
selected as a static modification. The variable modifications
allowed are methionine oxidation (+15.99492 Da) and acetylation of the N-terminus of the protein (+42.01057 Da).
Finally, the results are subjected to statistical analysis with the
Percolator algorithm to keep a false discovery rate (FDR)
below 1%.
3.5 Bioinformatic
Phase: In Silico Human
Gastrointestinal
Digestion
In a second step, the reference proteome is subjected to several in
silico bioinformatic analyses to predict potential bioactive peptides
encrypted in the parent sarcoplasmic fish proteome after an in silico
human gastrointestinal digestion.
1. Stomach: digest with pepsin all the proteins identified in the
discovery phase using the MS-Digest software which is
included in ProteinProspector v6.2.1 website. No missed cleavages were performed, and a minimum of 6 residues per peptide were selected as parameters.
2. Intestinal enzymes: digest sequentially the peptides obtained in
the stomach digestion (pepsin) with the intestinal enzymes
trypsin, chymotrypsin, elastase, carboxypeptidase A and B,
and aminopeptidases using the MS-Digest software, which is
included in the ProteinProspector v6.2.1 website (http://pros
pector.ucsf.edu/prospector/mshome.htm). No missed cleavages and a minimum of 6 residues per peptide were selected
as parameters (see Note 4).
3.6 Evaluation
of the Final List
of Potential Bioactive
Peptides
1. Rank the final list of the potential peptides using the PeptideRanker software that predicts, by employing the N-to-1 neural
network, the probability mark for the peptide bioactivity value
(http://bioware.ucd.ie/~testing/biowareweb/) [34]. Chose
the results with a score higher than 0.5 (!6 residues).
220
Mo ´ nica Carrera et al.
(1 μscans) using an isolation width of 3 amu and normalized
collision energy of 35%.
4. Fragmented masses are assigned during dynamic exclusion for
30 s after the second fragmentation event, and unassigned
charged ions are excluded from the MS/MS analysis.
3.4 MS Data
Processing
1. MS/MS spectra are analyzed using SEQUEST-HT™ (Proteome Discoverer 2.4 package, Thermo Fisher Scientific) against
the Teleostei UniProt/SwissProt protein database.
2. Use the following constraints for the searches: tryptic cleavage
with up to two missed cleavage sites and tolerances of 0.8 Da
for precursor ions and 0.6 Da for MS/MS fragment ions.
Carbamidomethylation (+57.021465 Da) on cysteine is
selected as a static modification. The variable modifications
allowed are methionine oxidation (+15.99492 Da) and acetylation of the N-terminus of the protein (+42.01057 Da).
Finally, the results are subjected to statistical analysis with the
Percolator algorithm to keep a false discovery rate (FDR)
below 1%.
3.5 Bioinformatic
Phase: In Silico Human
Gastrointestinal
Digestion
In a second step, the reference proteome is subjected to several in
silico bioinformatic analyses to predict potential bioactive peptides
encrypted in the parent sarcoplasmic fish proteome after an in silico
human gastrointestinal digestion.
1. Stomach: digest with pepsin all the proteins identified in the
discovery phase using the MS-Digest software which is
included in ProteinProspector v6.2.1 website. No missed cleavages were performed, and a minimum of 6 residues per peptide were selected as parameters.
2. Intestinal enzymes: digest sequentially the peptides obtained in
the stomach digestion (pepsin) with the intestinal enzymes
trypsin, chymotrypsin, elastase, carboxypeptidase A and B,
and aminopeptidases using the MS-Digest software, which is
included in the ProteinProspector v6.2.1 website (http://pros
pector.ucsf.edu/prospector/mshome.htm). No missed cleavages and a minimum of 6 residues per peptide were selected
as parameters (see Note 4).
3.6 Evaluation
of the Final List
of Potential Bioactive
Peptides
1. Rank the final list of the potential peptides using the PeptideRanker software that predicts, by employing the N-to-1 neural
network, the probability mark for the peptide bioactivity value
(http://bioware.ucd.ie/~testing/biowareweb/) [34]. Chose
the results with a score higher than 0.5 (!6 residues).
220
Mo ´ nica Carrera et al.