Byonic is uniquely capable of top- and middle-down searches, because it scores and annotates the original mass spectrum rather than a processed approximation. Byonic requires isotope-resolved precursors in order to determine precursor ion charges. Without isotope-resolved precursors, Byonic requires some user assistance to set precursor charges and masses.
Project Creation
Please refer to the below screenshots for a typical top-down search set up.
Instrument Parameters
Precursor mass tolerance: With isotope-resolved MS1, you can use a similar tolerance as you would use for bottom-up proteomics. Without isotope resolution, increase this value to a large number (for example, 10 Da) and enter precursor charge guesses on the Spectrum Input Options as described below. For top-down spectra without MS1, the precursor m/z recorded by the MS instrument is likely to be the center of the isolation window; in this case the tolerance may need to be very large (for example, 100 Da) in order to include the true precursor mass.
Digestion
Cleavage site(s): Leave empty for top-down searches
Cleavage side: N/A for top-down
Digestion specificity: Fully specific will only search for intact proteins (that is, complete FASTA file entries), semi specific will look for N or C terminal clips, and non-specific will look for internal fragments. Eg. If your protein has 200 amino acids, fully specific will search for a “peptide” spanning 1 – 200, semi specific will search for n – 200 and 1 – n for any choice of n, and non-specific will search for any peptide.
Missed cleavages: N/A
Modifications
The above example is an alkylated sample, hence carbamidomethyl is set as a fixed modification. If the sample is not reduced, include “Dehydro / -1.007825” modification to account for disulfide bonds; for speed set this modification as fixed rather than variable.
Spectrum Input Options
Max. precursor mass: Set this value higher than the largest protein mass in the sample.
Apply to unassigned spectra: Leave empty for isotope-resolved MS1. For non-resolved MS1, enter expected charge state numbers (E.g. 22,23,24)
Spectra Annotation (within the Project)
The default fragment ion annotation options in Byonic are set automatically based on the fragment mass tolerance setting. These may or may not be suitable for top-down results, due to the large number of fragments observed. Please refer to the screenshots below to set the spectra annotation option to “High/Low accuracy” or “Custom”.
Alternatively you can set custom annotation settings, with correction for isotope spacing as follows:
“Check isotope spacing” set to be on, and “Max number of peaks per 100 m/z” set to 100 or more will generally give good results. “Peaks” means individual peaks, not isotope clusters, so a limit of 100 peaks will translate to about 20 annotations per 100-Thomson stretch of the spectrum.
References
More details about the the Top-down searches with Byonic can be found here:
https://proteinmetrics.com/wp-content/uploads/2014/06/Bern.ASMS_.2014_Top.Down_.Final_.pdf