The Instrument parameters section sets values controlling precursor mass, fragmentation and recalibration. Both Dalton and ppm mass tolerances for precursors and fragments are supported, along with several fragmentation types
• Precursor mass tolerance sets the maximum mass difference between observed and theoretical (calculated) peptide mass.
• Fragmentation type sets the model(s) used to scan and score fragment peaks:
Internal models for most fragmentation types are included – CID low energy (ion trap), QTOF / HCD (beam-type CID), and ETD / ECD (electron transfer and electron capture dissociation), as well as a number of combinations of these types. These internal models determine which fragment peak types will be scored and annotated. For example, prominent c- and z-ions and small y-ions are expected for ETD. Prominent oxonium ions are expected from glycopeptides with QTOF / HCD fragmentation, but small or missing oxonium ions from CID low energy.
If a spectrum file contains more than one fragmentation type; for example, alternating HCD and ETD, Byonic will match the labels on the scans to the chosen fragmentation types, so that the MS/MS scans will be scored with the appropriate models. If a scan does not match the selected model, it will be ignored. For example, if the .raw file contains both HCD and ETD scans, the user must select “Both: HCD & ETD” or “Use Thermo scan headers” in order to score both types of scans with the correct model. Use Thermo scan headers is a recent option that supports all standard combinations of ETD, CID, and HCD, along with choices of mass analyzers.
If a spectrum file contains only a single fragmentation type, or, as in the case of *.mgf files, does not specify fragmentation type, then the user can choose any single fragmentation model for scoring. For example, HCD can be scored as CID low energy, or EThcD scored as pure ETD. Switching fragmentation models is occasionally advantageous, for example, when EThcD scans use extremely low collision energy.
For Sciex EAD fragmentation, use ECD.
• Fragment mass tolerance is the maximum difference in m/z for matching fragments. Byonic supports both relative (ppm) and absolute (Dalton) tolerances. Relative tolerances are generally recommended for time-of-flight and FTMS mass analyzers, and absolute tolerances for ion trap mass analysis. Fragment tolerances of 0.1 Da or 100 ppm or less apply to high-resolution MS/MS analysis, providing sufficient resolution to distinguish charge states of fragment ions. Fragment tolerances larger than 0.1 Da should be used with low-resolution (ion trap) MS/MS analysis.