Crosslink searches can be customized to accommodate for mixed linkages between different amino acids. Below is an overview describing the format and how to input the information in a search.
Case example:
Looking for a link between lysine (K) and asparagine (N); the crosslink results in loss of NH3 (17 Da)
Option 1: Search for any N-N and any K-K link in addition to N-K links.
This works well for purified proteins:
CustLink / -17.026549 @ K,N | xlink
Option 2: Search for any N-N links in addition to N-K links, but not K-K link.
- In the fasta file, replace each K with NJ. Leave each N as it is.
- Use the following modification rule:
KN-link / -17.026549 @ N | xlink - The nonstandard amino acid J is defined to have mass 100.00 Da, and K->N is -14 Da. To accommodate for this mass difference, we also need to add this adjustment in the modification fine control:
Fix-K-Remove-J / -100.00+14.05203 @ J | fixed
Option 3: Search selectively for N-N and K-K links in addition to N-K links.
For cases where the linkage region is known and the search shall only consider those, replace the corresponding K with KJ and N with NJ. J is defined to have mass 100.000, so you need to make a link:
KN-link / -217.02655 @ J | xlink
and also a variable modification like this to account for unlinked peptides:
Remove-J / -100.000 @ J | common3
Appendix:
Using a chemical crosslinker may result in dead ends. Dead ends may be added to your Xlink search as common modifications. For example a common chemical linker is DSSO.
The full DSSO custom entries are:
DSSO / 158.0038 @ K | xlink
DSSO / 176.0143 @ K | common2
C3H2O / 54.01056 @ K | common2
C3H4O2S / 103.9432 @ K | common2
C3H2SO / 84.982635 @ K | common2