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Filter to remove C terminal Arginine peptides

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Filter to remove C terminal Arginine peptides tcj11  2017-12-13 11:18
 
Hi, I was wondering how to
1)generate a filter to remove peptides with C terminal Arginine. Our heavy isotope control tissue only has lysines labeled, so I'd like to automatically remove all the R peptides, leaving only the K to more readily design SRMs.

Any ideas?

2) remove peptides with missed cleavages:
I tried K.*K|K*.K.*K and it didn't seem to work.

3) I also tried filtering to remove ragged ends, but this often seemed to remove some additional peptides:

ex A. - removed both 1. K.KVVFSTGSYPR.L (missed 1)
2. K.VVFSTGSYPR.L
other examples of peptides removed with ragged end filter
1. K.TWVLTPK.L
2. K.ADAHWITTPNR.M
3. R.EEIVYQWK.R
4. R.SSVEVGDTR.S

Thanks, Tija
 
 
tcj11 responded:  2017-12-13 11:22
Correction -
I think the string I used for peptide removal with missed cleavage worked fine.


Comments on 1) and 3) are welcome.
Thanks, Tija
 
Brendan MacLean responded:  2017-12-13 11:40
Hi Tija,
I suspect you will find if you look very closely that the ragged end filter is working correctly. Even the cases you highlight may be ragged end peptides. You haven't shown enough information to know for sure. You need to look at the protein sequence. For instance:

1. KK.TWVLTPK.L
2. KK.ADAHWITTPNR.M
3. RR.EEIVYQWK.R
4. RR.SSVEVGDTR.S

Could all be ragged end peptides if the AA before what you have presented is either K or R.

To filter for only peptides with N-terminal lysine, but add a new entry to the "Exclude peptides containing" list in Peptide Settings - Filter:

Name: Ending in R
Exclusion regular expression: R$

Make sure the entry is checked.

That should do it. Thanks for posting to the Skyline support board. Hope this helps achieve the target list you were hoping for.

--Brendan
 
tcj11 responded:  2017-12-13 20:08
Hi Brendan,
Thanks for that super helpful and rapid reply and the regular expression code. It is a hugely impressive software set!

Q1) you were right about the neighboring protein sequence and I have a follow up question - see attached ppt(I am just learning).

Q3) The Ending in R removal was great, but something odd happened with one extra peptide being removed that didn't have an R ending. see the attached example (possibly a user issue):)
Best regards, Tija
 
Brendan MacLean responded:  2017-12-14 18:28
Q1) It seems like you could use the regular expression "^K" to filter out anything starting with a Lysine, and that would give you what you are looking for?

Q3) I can't guess this one if the only difference you made in your settings was to change check an exclusion with the regular expression "R$". That makes no sense to me. I would have to guess that some other change occurred. If you feel sure it did not, then I will need you to post the file in question (using File > Share - Complete) to http://skyline.ms/files.url. Once it is there, let me know, and I will have a look.

Thanks for the screenshots and explanations.

--Brendan