In Standalone Byologic:
Add Chains in Protein input tab & build disulfide linkages in Disulfides tab
In the In-silico options Tab: Check Enable in-silico box> Use Table> Generate peptides by digesting proteins:
Select Disulfide Mode option: In this example we selected “Both” to add both crosslinked and free peptides. Add Mods and/or missed cleavages if desired. Click Generate:
An in-silico list is now made.
You can create project now in Byologic by clicking “Create” OR you can save and use this CSV in subsequent searches by: Right-click> Export to CSV (Figure a). Be sure to save this as unformatted (Figure b).
Important: The exported csv will NOT be in the correct format. You must delete the last 3 columns in the csv file to use in Byos searches.
Save newly made csv list of in-silico disulfides. To search, you can import this list into Byos under In-silico options (CSV import):
**Note: A pure in-silico search can be performed by right-clicking on the Byonic node’s header and deleting it.
Optional Step 1: There are advanced command that are particularlly useful for in-silico searches. Advanced commands may be entered during project creation in the Advanced configuration panel of the Advanced tab:
- Enable MS1 Correlation checks the MS1 isotope distribution. This is disabled (set to 0) by default.
- In-silico Align allows shifts in the start and end times to better match the signal. FeatureCenterTolerance specifies how much a feature (start and end time pair) may shift in minutes. FeatureDurationTolerance specifies how much a feature may grow or shrink in minute. For in-silico searches the value you place in both of these fields is the length of your chromatography in minutes. In this example, we have placed “60” for 60 minutes. You should adjust for your chromatography.
Optional Step 2: Add Shuffled Species: The Byologic table digest will only create “Expected” disulfide species. This is a manual workaround to add some “Shuffled” species.
In the Disulfides Tab: Delete the expected linkages from your Disulfide links (expected) table and add the shuffled species of interest:
Go back to the In-silico options tab: This time in the “Disulfide Mode” dropdown, choose “Disulfide Complexes only”. Click Generate:
You can then Export the table with this list of in-silico disulfide pairs.
If identified, the shuffled species will show as “Shuffled” in the Byos results: