The Database Manager supports isoforms and proteoform curation. By default, ProSightPD databases contain isoforms. If you want to expand to proteoforms, see (Optional) Expand Isoforms to Proteoforms. Editing your database at the isoform level, produces the largest proteoforms search space. This search space can be reduced by curating at the proteoform level. Thermo Fisher Scientific recommends that you annotate at the isoform level first, then expand and curate at the proteoform level. By editing at the isoform level, the database will be expanded at search time to include every possible proteoform of the isoform. It is easier to include a modification at the isoform level, expand the proteoforms, and remove selected proteoforms, than to expand the unmodified isoform into it's proteoforms and add all modified proteoforms one at a time.

IMPORTANT

Database expansion at search time is controlled by the Candidate Source parameter in the Subsequence Search node and the Annotated Search node. This parameter has the following four settings:

  • Isoforms
  • Proteoforms
  • Both - Exclude Isoforms with Proteoforms
  • Both - Include Isoforms with Proteoforms

In the ProSight Annotator dialog box, you can do the following:

  • Add or remove isoforms from a database.
  • Add or remove modifications from an isoform.
Figure ProSight Annotator view

Prerequisites

  1. You have the ProSightPD Database Manager open.

Procedure

  1. To edit or annotate the database, select ProSight Annotator from the ProSightPD Database Manager.
  2. To modify an isoform, do the following:
  3. From the Isoforms pane, select the isoform .
  4. From the residue pane, select the residue to modify .
  5. From the Point Features pane, select the modification that you want to apply.
  6. A colored square appears around the selected residue. The color matches the modification selected in the Point Features pane.
  7. (Optional) Lock the feature by selecting the lock next to the modification.

  8. NOTE

    After a modification is selected and locked, you cannot add additional modifications to the selected residue. The selected modification will be on all search candidates.

  9. To add a disulfide bond or endogenous cleavage:
  10. Select the plus icon, , in the Range Features pane.
  11. The Add Range Features dialog box opens.
  12. In the Add Range Feature dialog box, select the UniProt Feature Type and enter the Modification Start Point and End Point.
  13. Select the Save and Exit icon in the lower right corner.
  14. To add a mutation or cSNP (single-nucleotide polymorphism), select the residue to mutate.
  15. From the Point Features pane, select the cSNPs tab .
  16. Select the mutation.
  17. (Optional) In the protein sequence pane, you can add one or more modifications in the Point Features pane.

  18. TIP

    To remove a modification, hold down CTRL and select the modification. To select multiple point features, hold down the CTRL key.

  19. To add Custom Mass Modifications, select the residue to modify.
  20. 123456
    No.DescriptionNo.Description

    1

    Chemical Formula

    4

    Save and Exit

    2

    Mono Mass in Daltons

    5

    Custom modification

    3

    Residue

    6

    Green plus sign

  21. Select the Custom tab in the Point Features pane.
  22. Enter the Mono Mass in Daltons (Da) or the known Chemical Formula.
  23. Select the green plus icon to add the modification to the modifications list and apply the modification to the residue. If there is more than one Custom Mass Modification listed, you can select multiple modifications to add to each residue.

  24. NOTE

    Unlike named PTMs, the addition of multiple Custom Mass Modifications will result in orange colored squares.

  25. Select Save and Exit in the lower right corner.
  26. The downloaded database now appears in the list of searchable databases.