Multiple enzyme digestion
Closed this issue · 13 comments
Hi,
I have an experiment in which the samples (for specific reasons) have been digested using a combination of enzymes, LysC, Asp-N and Glu-N with 8 missed cleavages. Is there a possibility of enabling multi enzyme digestion to searchGUI?
Thank you for your help :)
You can set up your own enzymes via the Edit > Enzymes menu option in the main SearchGUI window. The created enzyme will then afterwards be available in the drop-down menu in the search settings.
I'm not sure how easy it is to combine c-terminal and n-terminal enzymes though and if any of the search engines are able to support this. But just give it a go and see what happens.
Thank you, I found it. :) I'll try it out!
It can't handle digestion from both N- and C- term, unfortunately. All amino acids gets defined as "cleaved after".
Ah, thanks for letting me know! There was a minor bug in our code that has now been fixed and a new version released (v4.1.23).
Still not sure how the individual search engines will deal with the combined modification though. But that I will leave for you to test. :)
I've quickly looked at the support for combining multiple enzymes for each of the search engines, in particular combinations of n- and c-terminal enzymes, and it may seem like X! Tandem (and maybe Andromeda) is the only one with explicit support for this. See the details for X! Tandem here: https://www.thegpm.org/TANDEM/api/pcs.html.
However, you would then have to define the enzymes individually and be able to select more than one in the search settings in SearchGUI, and this is not something we support in SearchGUI (nor in the downstream analysis in PeptideShaker) at the moment.
Supporting multiple enzymes is on our todo list, but I'm not sure when we will get the time to implement it.
Hi again. Seems like adding support for multiple enzyme selection in SearchGUI was easier than I thought! I think I now have a version working with X! Tandem. Needs a bit more testing before I'm confident to release a new version though, but I'll keep you posted.
Thank you for looking into it! I know andromeda allows this already but I'm not sure of how the other softwares handles it. I'm looking forward to the update then. :)
I just released SearchGUI v4.1.24 which adds support for selecting more than one enzyme in the search parameters dialog. It is currently supported by X! Tandem, Comet, MyriMatch and Andromeda (with Comet only supporting up to two enzymes).
While the searches with multiple enzymes all complete for the above search engines, I have not checked in details whether the results themselves make sense. I would therefore recommend looking closely at the results.
I've also confirmed that the search results can be opened in PeptideShaker, but there may be some side effects of using more than one enzyme that we haven't considered or been able to test. So please let us know if you come across any issues.
No problem, there seem to be some issues regarding the comet and X! Tandem searches.
There seem to be an loading issue with Comet.
And there also seem to be an issue with the target decoy concatenation of the database, when loading in peptideshaker.
There seem to be an loading issue with Comet.
But weren't you using a combination of three enzymes? Comet only supports up to two.
And there also seem to be an issue with the target decoy concatenation of the database, when loading in peptideshaker.
Seems like perhaps you have added some custom protein sequences to your database? Or rather non-standard headers? I'd recommend having a look at our Database help pages. In particular the section on non-standard headers: https://github.com/compomics/searchgui/wiki/DatabaseHelp#non-standard-fasta.
Alright, that would fix the problem. Thank you!
I didn't hear back from you in a while? Does that mean that this issue can be closed?
Yes, the issue is resolved. Thank you.