Import modifications to Skyline daily

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Import modifications to Skyline daily sandberg  2018-12-06
 

Hi,
I try to import a peptide search using Skyline daily. When I build the spectral library using a Mascot DAT file the program cannot find my modifications (Pic1) even though I can see that they are in the imported peptide list (Pic2). If I do the same thing in regular Skyline the modifications are found (Pic3). Is this a bug?

 
 
Brendan MacLean responded:  2018-12-06

Hi Magdelena,
Your pictures don't really agree with your description.

  • Pic1 - Skyline-daily is telling you that you don't currently have K[114] included in your Skyline document, but it knows of a Unimod modification with the name "GlyGly" that matches. You also don't have modification definitions for C[+23.03774] and M[+114.0293] but there are no matching modifications it knows of to choose from. You would have to define these yourself.
  • Pic2 - Skyline-daily is telling you that you have certain spectra which are being matched to multiple peptide sequences with scores above your cutoff. Because you did not choose the option to keep these ambiguous spectra in the library build form, they will be excluded from your library. Nothing about modifications in this message other than some of the spectra matching to modified peptide forms. This picture seems irrelevant to this support thread.
  • Pic3 - Your Skyline version shows 6 modifications not yet included in your Skyline document, but for which it can find definitions. I still doesn't know how to explain C[+23.03774]. But, it does have an explanation for "M[+114.0293]", which it gives as "2xCarbamidomethyl = M[114]". Since I can't find this in our Unimod definitions, I have to assume that it got defined through some other mechanism in your Skyline version, but not in Skyline-daily. If you define the same modification in Skyline-daily, then the two should behave the same with respect to the "M[+114.0293]" modification.

Hope this helps. I don't see anything that would imply a bug.

--Brendan

 
sandberg responded:  2018-12-06

Hi again Brendan,
Thank you for your answer and sorry for including irrelevant Pic 2. The problem is that I can see that the modifications are defined (Pic 4) but Skyline Daily still cannot find them (Pic1) in my Mascot DAT file while regular Skyline can find them (Pic 3).

 
Brendan MacLean responded:  2018-12-06

Sorry. What are you showing me in Pic4? I don't see "2xCarbomidomethyl" in that list. What do you mean by "the modifications". I think we are only talking about one modification that is different and that is "2xCarbomidomethyl", which I would expect to see in your list for Skyline but not Skyline-daily.

 
Brendan MacLean responded:  2018-12-06

Oh! Are you feeling that Skyline-daily is not aware of the other modifications that are listed when you use Skyline?

The imported search results appear to contain the modifications listed below.
Please select the ones you wish to add to the document.
2xCarbamidomethyl
Carbamidomethyl (K)
Carbamidomethyl (M)
Carbamidomethyl (N-term)
Cys->Dha (C)
GlyGly (K)

Of these 6, I believe the first is only defined (by your or in some way imported) in Skyline and not Skyline-daily. And the last behaves the same in both Skyline and Skyline-daily.

The middle 4 then are already included in your Skyline-daily document, which is why Skyline-daily is not asking you about adding them. This is borne out by Pic4.

I expect that if you show the same forms in Skyline as you did for Skyline-daily in Pic4 that you will find those 4 modifications are not checked in the Peptide Settings - Modifications tab, which is why Skyline is asking you about adding them to your document.

Sorry, that is so confusing. I guess we can think about improving the wording?

"The imported search results contain modifications not found in your document.
Please select the ones you wish to add to the document."

Would that have made more sense? Skyline doesn't bother telling you about the ones already explained by your document settings.

I hope this finally gets to the point of your question. Thanks for the screenshots.

--Brendan

 
sandberg responded:  2018-12-06

What I am saying is that when I import my mascot DAT file into Skyline Daily I would like the program to identify peptides from that file containing all these different modifications that you can see that I have defined in my peptide settings (Pic4) but still it can only identify one single modification which is GlyGly (Pic1). If I import the same mascot DAT file into regular Skyline the program can identify peptides containing many different modifications (Pic 3). What is wrong? How can I make Skyline Daily find all my modified peptides in the mascot DAT file?

 
sandberg responded:  2018-12-06

Ah, yes that is exactly what I meant and now I understand!

Thank you so much for this superfast support:D