Procedure

  1. Open a workflow template that includes the ProSightPD Annotated Proteoform Search node.
  2. If the workflow does not include the Annotated Proteoform Search node, drag and drop the Annotated Proteoform Search node into the workflow.
  3. Connect the node by dragging the arrows from the upstream node to the Annotated Proteoform node.
  4. Select the Annotated Proteoform node to view the parameters.
  5. Set the Input Database by selecting from the dropdown menu.
  6. NOTE

    To create a new database launch the Database Manager. To select a newly created database, you must close and reopen any open studies.

  7. Set the Precursor Mass Tolerance.
  8. The following are recommended values for narrow or wide tolerance searches:
  9. Narrow tolerance search is 2.2 Da
  10. Wide-tolerance search 200 Da
  11. NOTE

    For large modifications like signal peptides, consider setting your tolerance larger than the potential modification.

  12. Set the Fragment Mass tolerance.
  13. Thermo Fisher Scientific recommends 10 ppm. Use the narrowest tolerance possible for your data. As the tolerance widens, the search time and false positive rate increases.
  14. Set the FDR calculation.
  15. The default is True, which means that the application calculates the FDR based on the threshold set in the FDR node in the consensus workflow. To calculate the FDR, the FDR node must be present in the consensus workflow. If your sample is not complex or if you want to see all PrSMs, set this value to False to not calculate an FDR.
  16. NOTE

    Results are automatically displayed at the specified FDR cutoff for each result level (PrSM level, Proteoform level, Isoform level, and so on).

  17. (Optional) Select a static modification to apply to a specific residue.
    Currently, you can only apply one static modification in the search node. To set additional modifications, use the ProSight Annotator in the Database Manager.