Procedure
- Open a workflow template that includes the ProSightPD Subsequence Search node.
- If the workflow does not include the Subsequence Search node, drag and drop the Subsequence Search node into the workflow.
- Connect the node by dragging the arrows from the upstream node to the Subsequence Search node.
- Select the Subsequence Search node to view the parameters.
- Set the Input Database by selecting from the dropdown menu.
TIP
To create a new database, launch the Database Manager. To select a newly created database, you must close and reopen any open studies.
- Set the Precursor Mass Tolerance.
- Thermo Fisher Scientific recommends 10 ppm.
NOTE
Due to the large search space covered by the Subsequence Search, Thermo Fisher Scientific strongly suggests setting the precursor mass tolerance no greater than 25 ppm. For smaller data files or databases, you can use wider tolerances without causing the search time to increase.
- Set the Fragment Mass Tolerance.
- 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.
- Set the FDR calculation.
- The default is True, which means that the FDRs are calculated based on the threshold set in the FDR node in the consensus workflow. The FDR node in the consensus workflow is required. If your sample is not complex or you want to see all PrSMs, set this value to False to not calculate the FDR.
NOTE
Results are automatically displayed at the specified FDR cutoff for each result level (PrSM, Proteoform, Isoform, and so on).
- (Optional) Select a static modification to apply to a specific residue.
- You can apply one only static modification in the search node. To set additional modification, use the ProSight Annotator tool in the Database Manager.