In the Protein Sequence Editor, you can generate a list of possible proteoforms for a protein sequence and select which proteoforms to save with the protein sequence for Top Down Analysis.

Prerequisites

  1. You are editing a protein sequence in the Protein Sequence Editor.
  2. The sequence has the "Top Down" category for Top Down Analysis.

Procedure

  1. In the Protein Sequence Editor, expand the Site-Specific Variable Modifications for Top Down Analysis pane on the right side of the page.
  2. At the top of the pane, in the Number of Modifications per Proteoform boxes, enter the minimum (as low as 1) and maximum (as high as 3) modification sites for each proteoform.
  3. In the Select Type of Modification table, select the checkbox for a modification.
  4. To the right, the Define Site of Modification table automatically displays all the residue sites in the protein sequence that might have the selected modification.
  5. Select Type of Modification and Define Site of Modification tables
    Figure Select Type of Modification and Define Site of Modification tables
  6. In the Define Site of Modification table, select the checkbox(es) for the residue site(s) that you want to modify with the selected modification.
  7. Below the Select Type of Modification and Define Site of Modification tables, the Modification List table updates, and displays a working list of modifications. To clear all entries in the table, select Clear. To delete an entry in the table, select the modification row, and then select Delete.
  8. Modification List table
    Figure Modification List table
  9. Repeat steps 3 and 4 to add additional modifications to the protein sequence as needed.
  10. Modification List table with multiple added modifications
    Figure Modification List table with multiple added modifications
  11. When you are finished adding modifications, select the Generate Proteoforms button to the right of the Modification List table.
  12. At the bottom of the pane, the All Possible Proteoforms table displays the proteoforms generated from the Modifications List, along with the monoisotopic and average masses for each proteoform.

  13. TIP

    If you change the Number of Modifications per Proteoform range or edit the Modification List table, the application automatically clears the All Possible Proteoforms table.

    Select the Generate Proteoform button again to update the All Possible Proteoforms table.

  14. All Possible Proteoforms table
    Figure All Possible Proteoforms table
  15. In the All Possible Proteoforms table, select the checkboxes for the proteoforms that you want to save with the protein sequence for a search.
  16. When you select a proteoform in the All Possible Proteoforms table, the application highlights in green the associated modified residues in the Protein Sequence Map pane.
  17. (Optional) To save the unmodified protein sequence as a proteoform in addition to the generated proteoforms, select the Include Unmodified Sequence checkbox above the All Possible Proteoforms table.
  18. The unmodified sequence does not contain any variable modifications; however, it can contain previously assigned static modifications.
  19. When you include the unmodified sequence as a proteoform, it does not appear in the All Possible Proteoforms table.
  20. Save the protein sequence.