After the conclusion of the iPRG 2015 study, the raw data were reprocessed with Skyline 220.127.116.1119 by first creating spectral libraries with varying iProphet probability cut-offs: 0.95, 0.50, 0.15 and 0.05. A template document (included below) was created with similar settings to the original (Precursor charges 2, 3, 4; Max missed cleavages 2; All modifications from the search results), with the following changes on the Transition Settings – Full-Scan tab: Precursor mass analyzer “Centroided”; Mass accuracy ±10 ppm; Use only scans within 2 minutes of MS/MS IDs. A copy of this template was created with each of the 4 spectral libraries added. The iPRG2015.TargDecoy.fasta was imported into all 4 documents, followed by these 3 operations on the Edit > Refine menu: 1) Sort Proteins > By Name, 2) Remove Duplicates, and 3) Remove Empty Proteins. FDR was then assessed in the 4 files using decoy counting (see Supplementary Table 3 – FDR: No Duplicates). Each of the for 4 files was saved to a new name and Edit > Refine > Advanced – Min peptides per protein 2 applied. FDR was reassessed in the resulting files (see Supplementary Table 3 – FDR: Min 2 Pep) and the two estimates used to assess FDR in single-peptide proteins (FDR: Single Pep). The original raw data files were then imported and the “iPRG 2015” report associated with the documents exported. The report contains identical columns to the study SkylineIntensities.tsv file, minus the Probability and QValue columns. Despite the wider 4+ minute extractions windows, some truncated peaks remained, and their intensities were replaced with NA (missing at random). As before, the ions could be quantified by three isotopic peaks. The intensities of the isotopic peaks were summed (on the original scale) before the statistical analysis. Finally, we tested the impact of using only the monoisotopic peak for quantification, compared with this summing (see Supplementary Table 3).
Supplementary Table 3:
|iProphet cut‑off||No Duplicates||Min 2 Pep||Single‑Pep|
|Protein||Unique Pep||Protein||Unique Pep||Protein|
Exported MSstats Reports: