For top down analysis, you can assign site-specific variable modifications to a protein sequence to generate a list of proteoforms. The application saves the proteoforms along with the core protein sequence and uses them to identify fragment ions in the analysis.

Procedure

  1. Open the Protein Sequence Editor window by double-clicking Sequence Manager in the Experiment Types area on the BioPharma Finder home page.
  2. In the Sequence Manager, select a protein sequence by double-clicking the sequence name.

  3. NOTE

    For top down analysis, the protein sequence can only have one chain.

  4. The Protein Sequence Editor opens in a new window.
  5. In the Protein Sequence Editor window, on the left side of the page, select Top Down from the Category drop-down menu.

  6. NOTE

    If you change the experiment category of an existing protein sequence from Peptide Mapping, Intact Analysis, or Unknown to Top Down Analysis, the application will clear the variable modifications, disulfide bonds, and glycans previously applied to the sequence.

  7. On the right side of the page, expand the Site-Specific Variable Modifications for Top Down Analysis pane.
  8. Site-Specific Variable Modifications for Top Down Analysis pane
    Site-Specific Variable Modifications for Top Down Analysis pane
  9. In the Number of Modifications per Proteoform boxes, enter the minimum and maximum number of modification sites for each proteoform.
  10. The range that you enter determines how many proteoforms the application generates for the total list of proteoforms. You can then select from this total list which proteoforms you want to save with the protein sequence for a search.
  11. The minimum number of modifications per proteoform is one and the highest maximum number is three.
  12. In the Select Type of Modification table, select the checkbox for one variable modification.
  13. To the right, the Define Site of Modification table automatically displays the residue and amino acid information for all the sites in the protein sequence that can have the selected variable modification.
  14. Select Type of Modification and Define Site of Modification tables
    Select Type of Modification and Define Site of Modification tables
  15. In the Define Site of Modification table, select the checkboxes for the amino acid residue sites that you want to modify with the selected modification.
  16. Below the Select Type of Modification and Define Site of Modification tables, the Modification List table displays a working list of modifications used as input for generating the proteoforms. Scroll down as needed to view the entire list. To clear all entries in the table, select Clear. To delete a particular entry in the table, select that row, and then select Delete.
  17. Modification List table
    Modification List table

  18. NOTE

    If you define a variable modification but then assign it as a static modification, the application removes it from the list of variable modifications.

  19. Repeat steps 6 and 7 to add additional modifications to specific amino acid residues in the protein sequence.
  20. The Modification List table updates the working list of modifications as they are added.
  21. Modification List table appended
    Modification List table appended

  22. NOTE

    When you upgrade to BioPharma 5.2 from a prior software version, the Define Modification list is automatically reset; changes previously made to the default Define Modification list are not carried over.

    You must edit the Define Modification List to reinstate any custom modifications and/or user-specified default modifications (including glycans).