After viewing the results on the Process and Review page, you can use the real-time optimization feature to make changes to the processing method and deconvolution parameters and reprocess the experiment without leaving the Process and Review page.

Prerequisites

  1. For a top down analysis experiment, you have processed your experiment and are viewing the Process and Review page.

Procedure

  1. On the Process and Review page, expand the Real-Time Optimization title bar to view the Component Detection and Identification subtabs.
  2. The Component Detection pane expands and displays the peak-specific parameters used for processing the current experiment.
  3. On the Component Detection page of the Real-Time Optimization pane, select the Peaks, Peak # − Intact Fragmentation, and Peak # − Intact Deconvolution subtabs to update the corresponding parameters as needed.
  4. See Edit the Component Detection page.
  5. image/svg+xml Click here to see the subtabs if they are not already visible. Click here to reprocess with updated values.
    RTO pane (Component Detection page) showing the parameters under the Peaks subtab
  6. See Peak Selection parameters.
  7. image/svg+xml
    RTO pane (Component Detection page) showing the parameters under the Peak # − Intact Fragmentation subtab
  8. image/svg+xml
    RTO pane (Component Detection page) showing the parameters under the Peak # − Intact Deconvolution subtab
  9. See the following topics for more information on changing the Peak # − Intact Fragmentation and Peak # − Intact Deconvolution parameters:
  10. •ReSpect deconvolution parameters
  11. •Xtract deconvolution parameters
  12. Select the Identification tab and update the parameters as needed.
  13. See Edit the Identification page.
  14. image/svg+xml Click here to edit the protein sequences for target sequence matching.
    RTO pane (Identification page)
  15. The Sequence Matching Mass Tolerance and Multiconsensus Component Merge areas on the Identification subtab are active only if you select the Intact Deconvolution checkbox for at least one of the peaks under the Real Time Optimization > Component Detection > Peaks page because the application uses these parameters only for processing experiments containing full-scan MS data.
  16. To edit the protein sequence(s) added to the experiment for target sequence matching, select Edit at the upper right area of the Sequences Added to Experiment table.
  17. The Parameters > Identification page opens where you can add different sequences to the experiment or make other sequence-related changes as needed.
  18. See Edit the Identification page.

  19. NOTE

    When editing the protein sequence(s) using RTO, you can only change the modifications, glycosylation, or disulfide bonds information for a protein sequence.

    You cannot edit the protein chains.

  20. When you are finished editing the protein sequence(s), select the Process and Review tab to return to the Process and Review page for processing.
  21. If you add different sequences to the experiment, they appear in the Sequences Added to Experiment table

  22. NOTE

    If you change any protein sequence in the Sequences Added to Experiment table in the RTO pane, you must return to the Process and Review > Real Time Optimization > Component Detection page and reselect the protein sequence for each peak.

  23. When you are finished editing the Real-Time Optimization parameters, select Process in the command bar of the Process and Review page.
  24. In the Reprocess Experiment dialog box, enter the experiment and method names.
  25. image/svg+xml
    Reprocess Experiment dialog box
  26. You can choose to save the experiment under a new name or overwrite the existing experiment. You cannot overwrite an experiment that is currently submitted to the run queue or is actively processing or canceling.
  27. You can choose to save the method under a new name or overwrite the existing method (unless the method is a default processing method, which you cannot overwrite).
  28. For both the experiment name and method name, use only alphanumeric, space, underscore, and/or period characters.
  29. If the application finds invalid parameter entries, an error dialog box appears indicating the parameters that must be changed. To continue, enter the required parameter values within the appropriate ranges and select Process again to proceed.
  30. Select Reprocess.
  31. The application clears the previous results on the Process and Review page and submits a new processing job including your changes to the run queue.
  32. The RTO job moves to the top of the run queue, behind the currently running job. If no job is currently running, the application reprocesses the RTO immediately.
  33. While the new job is processing, you remain on the Process and Review page.
  34. When reprocessing is completed, the Process and Review page automatically displays the new results.
  35. For more information on the features and navigation of the Process and View page, see Viewing the Process and Review page for top down analysis.

  36. NOTE

    If you open another results file while the application is reprocessing your experiment, the application will not automatically update the display with the results of the reprocessed experiment.

    In this case, you can open your reprocessed experiment in the run queue by selecting the experiment name and selecting Open Results or by double-clicking the experiment name.