Peptide Identification from DDA and DIA

The DDA files were searched using Protein Pilot software v. 4.2 (AB SCIEX, Concord, ON, Canada) and Mascot v. 2.4 (Matrix Science Inc., Boston, MA, USA). The DIA files were converted to pseudo-MS/MS spectra with DIA-Umpire software and they were searched as DDA files [23 (link),24 (link)]. Trypsin as digestion enzyme was specified for both the software. For Mascot we used two missed cleavages, the instrument was set to ESI-QUAD-TOF, and the following modifications were specified for the search: carbamidomethyl cysteins as fixed modification and oxidized methionine as variable modification. A search tolerance of 0.08 Da was specified for the peptide mass tolerance, and 10 ppm for the MS/MS tolerance. The charges of the peptides to search for were set to 2 +, 3 +, and 4 +, and the search was set on monoisotopic mass. The UniProt Swiss-Prot reviewed database containing human proteins (version 2015.07.07, containing 42131 sequence entries) was used and a target-decoy database search was performed. False Discovery Rate was fixed at 1%.

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Butera G., Brandi J., Cavallini C., Scarpa A., Lawlor R.T., Scupoli M.T., Marengo E., Cecconi D., Manfredi M, & Donadelli M. (2020). The Mutant p53-Driven Secretome Has Oncogenic Functions in Pancreatic Ductal Adenocarcinoma Cells. Biomolecules, 10(6), 884.

Publication 2020

Protein Pilot software v. 4.2 Mascot v. 2.4

Cleavages Digestion Enzyme Human proteins Methionine Ms ms Peptides Protein Tolerance Trypsin

Corresponding Organization : Università degli Studi del Piemonte Orientale “Amedeo Avogadro”

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Variable analysis

independent variables

Protein search software (Protein Pilot v. 4.2 and Mascot v. 2.4)
Digestion enzyme (Trypsin)
Modifications (carbamidomethyl cysteins as fixed modification and oxidized methionine as variable modification)
Peptide mass tolerance (0.08 Da)
MS/MS tolerance (10 ppm)
Charges of peptides to search for (2+, 3+, 4+)
Database (UniProt Swiss-Prot reviewed database containing human proteins, version 2015.07.07)

dependent variables

Identified proteins

control variables

False Discovery Rate (fixed at 1%)
Instrument (ESI-QUAD-TOF)
Missed cleavages (two)
Search mode (target-decoy database search)

Annotations

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