3.8 MS Data
Processing
1. Process all the MS/MS raw files by the SEQUEST-HT algorithm (Proteome Discoverer 2.4 package) against a custommade database containing A. simplex s.s. plus Ascaris suum,
Toxocara canis, Brugia malayi, Loa loa, and Caenorhabditis
elegans UniProt/TrEMBL protein entries (see Note 33).
2. Use the following restrictions: full tryptic cleavage with up to
two missed cleavage sites and tolerances of 7 ppm Da for parent
ions and 0.6 Da for MS/MS fragment ions.
3. Set as fixed modifications: TMT-labeling (lysine and peptide
n-terminus) and carbamidomethylation of cysteine.
4. Set the permissible variable modifications: methionine oxidation and acetylation of the N-terminus of the protein.
5. Subject the results to statistical analysis to determine the peptide false discovery rate (FDR) using a decoy database and the
Target Decoy PSM Validator algorithm. Keep the FDR below
1%. Perform relative quantification using the Quantification
Module and normalization against total peptide amount (Proteome Discoverer 2.4 package).
6. Display a result statistics table. To obtain the final list of the
differentially expressed proteins apply several filters to the list:
(a) proteins quantified with at least two unique peptides, (b) at
least a twofold change between two types of samples (L3/L4
normalized ratios), (c) p-value 0.01 (Fig. 5b). Optionally
using data visualization mode create Volcano plots and PCA
charts of your result data (see ref. 5).
7. The total protein list or differentially expressed protein list can
be exported in Excel format for further analysis (in different
programs for statistics, visualization etc.).
8. Both protein lists can be submitted to the functional analysis in
Protein ANalysis THrough Evolutionary Relationships Classification System (PANTHER, http://pantherdb.org) and Database for Annotation, Visualization and Integrated Discovery
(DAVID 6.8, https://david.ncifcrf.gov/home.jsp) (Fig. 5c, d).
9. Additionally, proteins could be submitted to the network analysis to the Protein–Protein Interaction Networks Functional
Enrichment Analysis in STRING software (https://string-db.
org/) (see Note 34).
4 Notes
1. Use 1 M hydrochloric acid to adjust pH.
2. If using a glass beaker, the Tris can be dissolved faster if the
water is warmed to about 37
C, and the magnetic stir bar is
working immediately. Remember to bring the solution to
room temperature before adjusting pH.
Shotgun Proteomics for Anisakis simplex
71
Processing
1. Process all the MS/MS raw files by the SEQUEST-HT algorithm (Proteome Discoverer 2.4 package) against a custommade database containing A. simplex s.s. plus Ascaris suum,
Toxocara canis, Brugia malayi, Loa loa, and Caenorhabditis
elegans UniProt/TrEMBL protein entries (see Note 33).
2. Use the following restrictions: full tryptic cleavage with up to
two missed cleavage sites and tolerances of 7 ppm Da for parent
ions and 0.6 Da for MS/MS fragment ions.
3. Set as fixed modifications: TMT-labeling (lysine and peptide
n-terminus) and carbamidomethylation of cysteine.
4. Set the permissible variable modifications: methionine oxidation and acetylation of the N-terminus of the protein.
5. Subject the results to statistical analysis to determine the peptide false discovery rate (FDR) using a decoy database and the
Target Decoy PSM Validator algorithm. Keep the FDR below
1%. Perform relative quantification using the Quantification
Module and normalization against total peptide amount (Proteome Discoverer 2.4 package).
6. Display a result statistics table. To obtain the final list of the
differentially expressed proteins apply several filters to the list:
(a) proteins quantified with at least two unique peptides, (b) at
least a twofold change between two types of samples (L3/L4
normalized ratios), (c) p-value 0.01 (Fig. 5b). Optionally
using data visualization mode create Volcano plots and PCA
charts of your result data (see ref. 5).
7. The total protein list or differentially expressed protein list can
be exported in Excel format for further analysis (in different
programs for statistics, visualization etc.).
8. Both protein lists can be submitted to the functional analysis in
Protein ANalysis THrough Evolutionary Relationships Classification System (PANTHER, http://pantherdb.org) and Database for Annotation, Visualization and Integrated Discovery
(DAVID 6.8, https://david.ncifcrf.gov/home.jsp) (Fig. 5c, d).
9. Additionally, proteins could be submitted to the network analysis to the Protein–Protein Interaction Networks Functional
Enrichment Analysis in STRING software (https://string-db.
org/) (see Note 34).
4 Notes
1. Use 1 M hydrochloric acid to adjust pH.
2. If using a glass beaker, the Tris can be dissolved faster if the
water is warmed to about 37
C, and the magnetic stir bar is
working immediately. Remember to bring the solution to
room temperature before adjusting pH.
Shotgun Proteomics for Anisakis simplex
71