The Targeted Top-Down workflow

The Targeted Top-Down workflow is designed to look for specific proteoforms of interest in a spectrum using TDValidator.

The Targeted Top-Down workflow is partitioned into the Results and TDValidator tabs.

Create a Targeted Top-Down workflow

Procedure

  1. From the Home screen, select the Targeted Top-Down workflow.
  2. Navigate to the folder containing the desired file, select it, and select Open.
  3. Select the TDValidator > Fragmentation tabs, then adjust the Calibration and Scan Range.
  4. Select an option from the Scan Filter dropdown.
  5. Select an appropriate option from the Fragmentation dropdown list (for example, CID, HCD, or ETD).
  6. Select the TDValidator > Precursor tab, then adjust Calibration and Scan Range for the precursor peak.
  7. Select Input Spectrum in the bottom-right corner.
  8. Select Fixed Modifications, adjust the Cysteine and Methionine modifications, then select Save.
  9. Select Options, adjust the settings if needed, then select Save.
    Figure 1. TDValidator Settings

    TDValidator Settings
  10. Perform one of the following:

    • Select Add Manually, name the proteoform, fill in the sequence, and select Save.

    • Select Add from fastp, navigate to the folder containing the desired proteoform, select it, then select Open.

    • Select Add from Databases, select the desired database or create a new one, then adjust the variable features, and select Expand.

  11. In the Precursor tab, review the Matching Precursor Ions table and the Precursor Spectrum.
  12. In the Fragmentation tab, review the Fragment Map, as well as the following:

    • Matching Fragment Ions to see fragment ions grouped across the spectrum.

    • Matching Fragment Mass Groups to see matched fragment masses grouped across charge states.

    • Theoretical Ions to inspect theoretical fragment ions generated from the selected proteoform.

    • THRASH Ions when AIM annotations are enabled.

    • Xtract Ions when AIM annotations are enabled.

  13. Review the m/z Spectrum and Mass Spectrum.
  14. Select Ion Intensities to compare proteoforms, review Residue Intensities, Terminus Ion Intensities, Residue Cleavages, Filter Scores, and Mass vs Intensity statistics.
  15. Select Metrics to review the S/N Histogram, S/N vs Score, PPM Charge Map, PPM Differences, Mass Shifts, Ion Mapping, and Fragment Locations.
  16. Select the Decoy Analytics tab and enter the number of decoy runs to perform and select the proteoform to analyze.
  17. Select the Start/Resume FDR Calculations On the Proteoform Selected Below button GUID-8E6B5452-8680-4AFD-972D-6AB90C31E856-print.png to start or resume decoy calculations, select the Stop FDR Run button GUID-75FD1BA4-34EC-4692-AF03-03ADFACD68F1-print.png to pause the current run if needed, select the Add More Decoy Runs button GUID-6513E813-70CE-4BC8-AC89-200E38EAFC10-print.png to add additional decoy runs, and select the Reset FDR Results button GUID-524CEDDE-9590-45C6-BFD2-0481747D762C-print.png to clear the existing decoy runs.
  18. Review the Matching Ions distribution and Sequence Coverage.
  19. Select Export, navigate to the desired folder, and select Save to save decoy results in .csv format.
  20. Select Save Proteoforms and select one of the following from the dropdown list:

    • Select As .fastp File then select whether to export all proteoforms or only proteoforms with matched precursor ions, matched fragment ions, or both.

    • Select As ProSight Database then select whether to export all proteoforms or only proteoforms with matched precursor ions, matched fragment ions, or both.

  21. Navigate to the desired folder, name the file, and select Save.
  22. Select Save to TDV to preserve the complete analysis state, navigate to the desired folder, name the file, and select Save.