Byonic gives the user detailed control of the types and numbers of posttranslational
modifications allowed on candidate peptides. Modification Fine Control™ enables Byonic
to focus its search on the candidate peptides most likely to match mass spectra, so that
with the right parameter settings, Byonic can find more true matches more quickly.
One of the most commonly asked questions is the definition and the difference between
“common” and “rare” type variable modifications in Byonic. The simplest way to think
about them are “variable modification type A” and “variable modification type B”. In a
majority of analyses, completely swapping common and rare designations of modification
make no difference in the results. Hence, one does not need to know in advance which
modifications are commonly occurring in the sample. What is important are the total
common/rare max values, and to keep in mind that combinations of common and rare
type of modifications are allowed.
A simple set of modifications are as follows:
Here, oxidation and deamidation are set as rare1, which means a peptide can only be
identified with one oxidation (on M or W), or with one deamidation (on N or Q). A peptide
that contains both modifications concurrently will not be identified. Gln->pyroGlu is set as
common, because we expect protein N-term Q residues to be mostly cyclized to pyroGlu.
Hence, when performing PTM analysis, we would like to identify peptides which
concurrently contain pyroGlu and an oxidized M. Ideally, the goal should be to find the
balanced settings that allow reasonable combinations, but does not include so many
options that will significantly slow the search or introduce the possibility of false positive
identifications.
To demonstrate further, please take a look at the table below. In each row, we change 1
parameter (highlighted), and present the peptides that can be identified with these search
settings:
| Variable Modifications | Total common/rare max | Peptides Identified | Comments |
| Oxidation / +15.994915 @ M, W | rare1 | 1/1 | QMMNQ QMMNQ QMMNQ |
|
| Oxidation / +15.994915 @ M, W | rare2 | 1/1 | QMMNQ QMMNQ QMMNQ |
Total rare max = 1 sets the restriction, so rare2 gives same results as rare1 above. |
| Oxidation / +15.994915 @ M, W | rare2 | 1/2 | QMMNQ QMMNQ QMMNQ QMMNQ |
Restriction is removed. Doubly modified peptide can be identified. |
| Oxidation / +15.994915 @ M, W | rare1 Gln->pyro-Glu / -17.026549 @ N-Term Q | rare1 |
1/1 | QMMNQ QMMNQ QMMNQ QMMNQ |
Total rare max = 1 allows only one rare type modification per peptide. |
| Oxidation / +15.994915 @ M, W | rare1 Gln->pyro-Glu / -17.026549 @ N-Term Q |common1 |
1/1 | QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ |
Cross-category combinations are allowed. Singly and doubly modified peptides can be identified. |
| Oxidation / +15.994915 @ M, W | rare1 Deamidated / +0.984016 @ N, Q | rare1 Gln->pyro-Glu / -17.026549 @ N-Term Q | common1 |
1/1 | QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ |
Adding one more modification. Here pyroGlu and Deamidated Glu are colored differently for clarity. |
| Oxidation / +15.994915 @ M, W | rare1 Deamidated / +0.984016 @ N, Q | rare1 Gln->pyro-Glu / -17.026549 @ N-Term Q | common1 |
1/2 | QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ QMMNQ |
This row demonstrates how increasing total max values changes the allowed list of modification combinations. |
Please see other application notes, videos and other resources for more information on modification fine control.