The Byos De Novo Sequencing (also known as Supernovo) default workflow requires little adjustment to processing parameters compared to other Byos workflows. Only three processing parameters may be defined by the user:
- Fragmentation type
- MS1 and MS2 mass tolerances
- Cysteine alkylation
These processing parameters are defined by a .byparms file (Byonic Parameter File).
By default, Byos De Novo Sequencing uses a .byparms file set for HCD or CID fragmentation, typical MS1/MS2 tolerances for modern high resolution mass spectrometers, and iodoacetamide alkylation. Oftentimes the default settings are suitable. However, different alkylating agents (like iodoacetic acid) and fragmentation types (like EThcD, or HCD+EThcD) require an Alternative Byonic Parameter File be specified in the Byos De Novo Sequencing workflow processing nodes tab.
The .byparms files Default and Cysteine58 can be found in the Program Files>ProteinMetrics>PMI-Suite>Tools>SupernovoCommandLine folder. Default is used when Alternative Byonic Parameter File is left blank. Cysteine58 is the same as Default, except it is set up for iodoacetic alkylation instead of iodoacetamide (+58 vs. +57 fixed modification on Cysteine). If needed, Alternative Byonic Parameter File is used to direct the workflow to use the Cysteine58.byparms file instead of the Default.
Creating and loading a new .byparms:
Sometimes a new .byparms file must be created, typically to support different fragmentation types. Follow the steps below to create and link to a new .byparms file.
1. Open the Default .byparms file.
2. Define new .byparms settings as needed, using the Default as a starting point. Only adjust the instrument parameters and fixed Cysteine modification.
2a. Instrument Parameters: Precursor mass tolerance of 10 ppm is almost always suggested. The Fragmentation type should be defined. By Default QTOF / HCD is selected, which is suitable for CID (including HCD fragmentation). Other common fragmentation types used for de novo sequencing include ETD and EThcD. Multiple fragmentation types can be processed at once and defined as the Fragmentation type. One common example for de novo sequencing is Both: HCD & EThcD, which is suitable for both HCD/CID and EThcD MS/MS spectra. If data is acquired using Thermo instrumentation, the Use thermo scan headers Fragmentation type can be selected. As the name implies, Byos will automatically read Thermo scan headers and process different fragmentation types accordingly. Once the Fragment type is defined, the Fragment mass tolerances should be set accordingly.
2b. Modifications: The cysteine fixed modification can be adjusted as needed (see figure below). All other modifications must be left as is.
3. Click File>Save params... and select a folder and name for the new .byparms file. Make note of where the new .byparms file is saved.
4. Open the De Novo Sequencing workflow in Byos and go to the Processing nodes tab. Select the ... button for Alternative Byonic Parameter File and browse to the newly created .byparms file.
The workflow is now set up with a new Alternative Byonic Parameter File and can be saved as a .wflw for future use.