Selecting for a structural modification

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Selecting for a structural modification sstoychev  2018-08-09 03:11
 

Hello,

What will be the best way of selecting for a structural modification. For example, I'm interested in seeing how all N-terminal acetylated peptides are behaving in the samples. I know how to select these in Skyline (Peptide settings - Modifications) but how would you remove all other peptide sequences i.e. other than N-terminal peptide. Ideally I would also want to see ratio of acetylated vs non-acetylated for same peptide if possible, if not looking at levels of acetylation between samples will be sufficient.

Perhaps the Edit - Exclude peptides (not matching expression) will allow for selection of N-terminal acetylated peptides only?

Stoyan

 
 
sstoychev responded:  2018-08-09 05:53

The solution I have found thus far is to leave all matched peptides in the list, perform MS Stats on all peptides and filter these based on "Peptide Modified sequence" with filter being "contains [+42], this will select all acetylated peptides in the list (not just N-terminal) but narrows down list to a workable size for manual checking of the data

 
Brendan MacLean responded:  2018-08-09 08:50

Hi Stoyan,
The Peptide Settings - Filter tab also has a solution in the "Exclude peptides containing" list. If you add one of these filters, you will find that it is possible to create a double negative and "Exclude peptides NOT containing" (by choosing the "Not matching expression" option). Then you just have to come up with a regular expression matching what you want to filter for.

You will also want to choose the "Light modified sequence" option when trying to match a modification and then you will want to be aware that brackets ([ and ]) and plus (+) are special characters in regular expressions and therefore need to be escaped. So, you might use the following expression:

^.\[\+42

to match "Acetyl (N-term)".

It worked for me anyway. Hope this helps.

--Brendan

 
sstoychev responded:  2018-08-10 05:23

Thanks Brendan,

I tried your suggestion but it did not filter any of the existing peptides. I have attached a screenshot of the settings. I assume with these settings only peptide containing +42 (acetyl mod) will be selected and everything else removed?

Also, what does the Applied to Light modified sequence" option do? In this case would it select for the peptides that can contain +42 but are not modified? So one can than look light : heavy ratio i.e. peptide with and without acetylation.

 
Brendan MacLean responded:  2018-08-11 08:58
It looks like the support page translation for "Markdown" stripped critical backslash characters. Also, don't add the closing bracket the way you did, because the full text is probably "[+42.0]". I have changed the "Render As" property of this response to "Plain Text" and hope that will send you the full text of my proposed filter:

^.\[\+42

But, I have also included a screenshot of what you are aiming for and what works for me. (I also corrected the Markdown version above.)
 
sstoychev responded:  2018-08-12 01:35
still does not filter. I've set it up as per attached.
 
Brendan MacLean responded:  2018-08-12 07:14
Maybe start with a new document and confirm for yourself that this works when you import new FASTA. It definitely works for me. Maybe confirm for yourself that any of the existing filters work for a new document with new FASTA.

For your own document, if you have applied any manual edits that could keep Skyline from applying the filter. And you might need to use Edit > Refine > Advanced - Auto-select all: Peptides to force Skyline to use the settings and filter you have chosen. But, that would also override any manual edits you had made.

You could also export the ModifiedPeptideSequence field for all of the peptides in your document, use whatever extern software you like to filter that down to just the peptides you want for any reason you want, and then just use Edit > Refine > Accept Peptides to reduce your targets to just those.

Anyway, if you want to use an "Exclude peptides containing" filter, first convince yourself you can get any such filter to work, and then convince yourself that the filter I have suggested works as advertised, and then figure out why your document is blocking the filtering.

Good luck. Sorry, this hasn't gone more smoothly.

--Brendan