No cleavage if variable modification

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No cleavage if variable modification dawn dufield  2018-11-13
 

HI Brendan,

I was wondering if there is a way to adjust the cleavage settings on an enzyme or define a new enzyme that would NOT cut when there was a variable modification ... ie if something were to modify lysines could you have skyline no longer cleave at that lysine automatically or do you need to somehow add them all manually? or with partials?

 
 
Brendan MacLean responded:  2018-11-14

You can achieve this by relaxing your limit on missed cleavages, and then adding an exclusion in the Peptide Settings - Filter tab excluding unmodified missed cleavages.

Suppose you want no unmodified missed cleavages. You could relax that to 5 and then add the following exclusion:

Exclusion regular expression:
K.*K

Apply match to:
(o) Light modified sequence

Exclude peptides:
(o) Matching expression

I just did this with two yeast proteins and the modification "Acetyl (K)". With just 5 missed cleavages and this variable modification I had 4,329 peptide targets. With the exclusion that is reduced to 444 peptides.

Hope this helps.

--Brendan

 
dawn dufield responded:  2018-11-14

Hi Brendan,

This seemed to maybe work for chymo, but did not work for Trypsin. Essentially I want Trp to cut at every K and R unless the K is either followed by a P or is modified. I have the lysine set to variable modification, but if I up the missed cleavages and follow your instructions I seem to get a bunch of missed R and that is not what I need. is there a way to tell it to cut at every R and every K unless it is a modified K?

Thanks
Dawn

 
Brendan MacLean responded:  2018-11-15

You should change the regular expression I supplied to:

K[^[]|R.|K[^A-Z]+$

Which excludes:

  1. K followed by any character but [ which starts a modification description
  2. R followed by any character (i.e. only R at the C-terminus is allowed)
  3. K followed by only non-A-Z to the end of the text (i.e. K followed by a modification description only is not allowed at the C-terminus)

Clearly, I underestimated the complexity of getting this exclusion to match your needs, but I hope this one will work for you.