filter peptide list for modifications at specific amino acids

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filter peptide list for modifications at specific amino acids kenhsu  2018-01-08 09:42
 
Hello,

We are interested identifying peptides with particular modifications on amino acids from lists that contain both modified and unmodified peptides.

What is the best strategy?

Thank you.
 
 
Brendan MacLean responded:  2018-01-08 11:19
Skyline has the ability to do this for you when you are starting with proteins. You can just use the Peptide Settings - Filter tab "Exclude peptides containing" list.

Click Edit List.
Click Add.
Give your exclusion a name.
Make Apply match to = Light modified sequence
Make Exclude peptides = Not matching expression
Then specify a regular expression matching what you want to keep.

For example someone interested in phospho modifications on S and T could use the expression:

[ST]\[

In fact, if your case is as simple as wanting to include any modification on certain amino acids, you can just extend the expression above:

[STQC]\[

Save it and check it in the Peptide Settings - Filter tab and you should have your modification filter for target proteins.

Hope this is helpful.

--Brendan
 
kenhsu responded:  2018-01-09 10:14
Thank you Brendan.

Just to confirm - we enter the mass for the modification in the example expression, e.g. [STQC]\[483]
 
Brendan MacLean responded:  2018-01-09 11:04
No, that is not correct. You don't need to put the mass unless you expect there to be other types of modifications on the same amino acid but with different masses, and what you put is not even good regular expression syntax. (see here https://www.regular-expressions.info/reference.html)

Either you use exactly what I proposed:

[STQC]\[

Or, if you want to use masses, then you would specify them like this:

[ST]\[\+80\]|C\[\+57\]|Q\[-17\]

But, that seemed pretty unlikely to me, since Skyline already lets you choose which modifications to use. So, why would you choose modifications just to exclude them?

Up to you, though. Hope this helps.

--Brendan
 
kenhsu responded:  2018-01-09 11:10
We are developing probe molecules that covalently modify different amino acids. Depending on the probe molecule tested, the mass adduct and amino acid modified will vary. Thus, we need a way to filter our large datasets for specific modifications to identify targets of our small molecule probes.

I hope this clarifies our goal for the filtering step.
 
Brendan MacLean responded:  2018-01-09 11:13
Okay. Will what I have explained work for you now?
 
kenhsu responded:  2018-01-09 12:34
Does not seem to be working with either [STQC]\[ or [ST]\[\+80\]|C\[\+57\]|Q\[-17\] approach.

The number of proteins and peptides shown in the Skyline does not change before vs after the filter in peptide settings?
 
Brendan MacLean responded:  2018-01-09 13:16
How did you add your target peptides? As a peptide list? Or by adding protein sequences?

If the latter and this isn't working, then you can try Edit > Refine > Advanced - Auto-select all: Peptides (checked).

That really should work for protein sequences. If not, then something must be wrong in your exclusion.

If you used peptide lists, then you may need to pre-filter yourself, or re-add your peptides.

Feel free to post an example using File > Share to create a ZIP file (ideally not a large one) and attach that to your next response.
 
kenhsu responded:  2018-01-09 19:35
Okay, figured out the problem. After the filtering as you suggested, you need to change Library/pick peptides matching to "library and filter."

New problem: how do I keep probe-modified light and heavy peptides in SILAC samples?

Thanks!
 
Brendan MacLean responded:  2018-01-09 19:58
Ah. Good point! Nice troubleshooting.

I don't understand the new questions. Can you clarify? Maybe a screenshot?
 
kenhsu responded:  2018-01-10 05:51
Before applying the library and filter step, I now see chromatogram information unavailable for the modified peptides that remain.

Before the filter step, I can see the light and heavy version of each peptide.

Not sure how to filter and still view the chromatograms for both light and heavy probe-modified peptides.
 
Brendan MacLean responded:  2018-01-10 06:26
Please provide your document using File > Share complete to create a single ZIP file, and post that to

http://skyline.ms/files.url

Let me know when it is there. Thanks.