Filtering Fragmentation Spectra
If you have a complex data set including fragmentation spectra of peptides and glycopeptides but are only interested in the identification and/or quantification of glycopeptides, the runtime can be decreased significantly by applying MSMS Filtering in the Byonic search settings.
The input window for Peak Filters provides a dropdown list of common diagnostic peaks that can be extended with custom peaks.
Depending on the mass accuracy settings of the instrument, the m/z tolerance can be defined in Dalton. Required Number sets the number of diagnostic peaks required in the fragmentation spectra to be included. If the number is set to 0, no MS/MS Filtering will be applied.
Applying the "isobar_score_filter" advanced Command
As of v5.1, Protein Metrics software has also addressed the potentially long search times for O-linked glycosylation by using the shared peptide sequence of multiple possibilities to optimize the search space. Even a modest search of 8 proteins against 9 glycan compositions, allowing a maximum of 2 glycan modifications per peptide, can result in hundreds of millions of glycopeptide candidates. We used an intelligent scoring mechanism for ‘isobaric peptidoforms’ that does not waste time looking for related yet poorly-scoring candidates if they have already been identified.
Figure 1: Peptidoforms
A more detailed description can be found here:
Briefly, in the above mentioned source, the assumption was validated that within a group of isobaric peptidoforms scored against the same spectra, no candidate will score significantly higher or lower than another. Hence, the total search speed-ups by omitting the scoring of subsequent isobaric peptidoforms against a set of spectra, if the first form scored does not reach a threshold score.
Figure 2: Case Examples
This logic can be turned on during Byonic processing by using the following advanced command:
isobar_score_filter=50
where 50 is the threshold score to allow subsequent candidates to be scored. We suggest using a filter of 50, which is empirically chosen, to achieve faster processing times without losing sensitivity.
As a reminder, Byonic advanced commands are entered in the custom modifications box as follows:
Example results:
| Filter Off | Filter On |