In Byos, under the PTM workflows, you can adjust the protease specificity to accommodate any custom protease used in your experiment.
There are two places to input this information: Under the (1) Samples tab and in the (2) Byonic node. Below is a description of each.
1. The Samples Tab: Inputting your proteolytic information enables missed cleavage calculations and related functionally in the reports. This setting does not impact your search and is optional.
1a. To change one sample at a time, simply double-click in the digestion column and select the protease:
1b. To change multiple samples at a time, shift select and right-click to open the popup:
Bulk editing non-neighboring rows: Hold the Ctrl key while selecting the rows. Then, right-click and select Edit selected row(s).
1c. Custom proteases: Customization of this menu is also possible. Simply type in a name and the unique protease sites. Note, the format of the text must not change:
In the example above, the NewProtease has C-terminal cleavage sites at proline, methionine, and lysine.
The format is as follows: NewProtease @ P,M,L | C-term
2. The Byonic Node: Inputting your proteolytic information controls the Byonic search space used to identify your peptides. This setting dictates your search and is required (except for non-specific searches).
2a. In the example below, we are performing a search based off of a tryptic digest:
In the search above, we will be looking to identify peptide that have cleavages C-terminal to R and K. We are also allowing for 2 missed cleavages. Note: Byonic does search for a K/P cleavage, even when set to fully specific. However, it doesn't count K/P as a missed cleavage.
2b. General guidance for the most common enzymes can be taken from the samples tab drop-down as shown in picture 1b. The text is as follows:
Trypsin: R, K @ C-term
Chymotrypsin: W, F, L, Y @ C-term
Thermolysin: A, F, I, L, M, V @ N-term
LysC: K @ C-term
GluC: D, E @ C-term
AspN: D @ N-term
LysN: K @ N-term
ArgC: R @ C-term
2c. Customization of the Byonic tab is also possible. To do this, simply input the protease cleavage site(s):
2d. Lastly, cleavage sites from both termini are supported. To input this information, simply input the amino acids as "C-term;N-term" as shown below: