The search results are filtered by DTASelect. This is performed automatically after the search and can be repeated to adjust the filtering parameters. The primary options for this are shown in the following window and steps. Further options can be found by clicking on the “Additional DTASelect options” link and a general description can be found in a Current Protocols in Bioinformatics chapter.[Cociorva Ref] The following options are generally best for phosphoproteomics.

  • Make the appropriate “Basic DTASelect 2.0 Parameters” selection for phosphoproteomics.
    1. Enter “1” for “Minimum number of peptides per protein (-p)”. Only one phosphorylation site or phosphopeptide may be present or detected for a protein.
    2. Select “1” for “Minimum number of tryptic ends per peptide (-y)”. If a “Specificity” of “none” was used in the search, “0” can also be selected to maximize phosphopeptide identifications.
    3. Enter a desired “False positive rate (—fp)”. A common FDR is 0.1% at the peptide level.
    4. Enter a “Precursor delta mass cutoff (-DM)”. A common value is 10 ppm for Orbitrap data, but can be assessed based on the precision of the instrument used.
  • Make the appropriate “Advanced DTASelect 2.0 Parameters” selection for phosphoproteomics.
    1. Select a “Peptide modification requirement” of “1”.
    2. Select “yes” for “Statistics with delta mass (—mass)”.
    3. Select “yes” for “Statistics with modifications (—modstat)”.
    4. Select “yes” for “Statistics with tryptic status (—trypstat)”.
    5. Leave the default of “Both” for “Include heavy search” unless only the unlabeled “Light only” or isotopically-labeled “Heavy only” peptides and proteins need to be identified or quantified.
    6. Enter other advanced options as necessary in “Protein ID filter (-e)”, “Peptide sequence filter (-Sic)”, and “Additional DTASelect options”.
    7. Select “Overwrite the previous” if the previous DTASelect result is unwanted or “Run as new” if the previous DTASelect result is wanted.

Figure 9.3.1: DTASelect

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