You selected a processing workflow in the New Study and Analysis dialog box. In this section, you configure the node parameters.

Procedure

  1. Select the Workflows tab.
  2. In the Analysis pane, select Edit on the Processing Step.
  4. Select the Spectrum Files RC node in the Workflow Tree pane.
  5. For the Protein Database parameter, select Escherichia coli (strain K12) (sp_canonical TaxID=83333_and_subtaxonomies) (v2022-04-30).fasta in the parameters pane.
  6. The E. coli FASTA file is the one you obtained in Add the FASTA file to Proteome Discoverer.
  7. For the Dynamic Modification parameter, select Label: 13C(6)/+6.020 Da (R).

  8. NOTE

    The Spectrum Files RC needs only to identify sufficient peptides to perform the mass correction. We chose to include only the medium label for arginine as a variable modification—while excluding the other forms of lysine and arginine. The node finishes significantly faster when using fewer modifications. The Spectrum Files RC node uses a Sequest HT search to identify peptides to use for mass recalibration.

  10. Select the Sequest HT node in the Workflow Tree pane.
  11. For the Protein Database parameter, select Escherichia coli (strain K12) (sp_canonical TaxID=83333_and_subtaxonomies) (v2022-04-30).fasta.
  12. The E. coli FASTA file is the one you obtained in Add the FASTA file to Proteome Discoverer.
  13. For the Fragment Mass Tolerance parameter, change the value to 0.02 Da.

  14. NOTE

    The default, 0.6 Da, is appropriate for CID or HCD spectra acquired in the ion trap mass spectrometer on a hybrid instrument. Because these MS/MS spectra were acquired in the Orbitrap mass analyzer, 0.02 Da is a more appropriate value.

  16. For the Sequest HT node’s Dynamic Modifications parameters, keep Oxidized (M) and select each SILAC form as modifications in the Sequest search.
  17. Label:2H(4) (K)
  18. This is the medium SILAC label for lysine.
  19. Label:13C(6)15N(2) (K)
  20. This is the heavy SILAC label for lysine.
  21. Label:13C(6) (R)
  22. This is the medium SILAC label for arginine.
  23. Label:13C(6)15N(4) (R)
  24. This is the heavy SILAC label for arginine.
  25. The processing workflow is complete.