Prosit .msp library import is missing iRT values anatoly.urisman  2020-04-25
 

Hi Skyline Team,
I was wondering if the iRT values in Prosit generated .msp libraries could be parsed upon import into Skyline. I use a simple conversion script as a work-around, but this adds an extra step. I suppose another solution would be to change Prosit output to Skyline-readable.
Thanks!
Anatoly

Here is an example of Prosit .msp format...

Name: TIFGGSDSTNQGPSNGER/2
MW: 912.41136795554
Comment: Parent=912.41136795554 Collision_energy=28.0 Mods=0 ModString= TIFGGSDSTNQGPSNGER///2 iRT=32.720001220703125
Num peaks: 46
175.118952167 0.10104765714257913 "y1/0.0ppm"

 
 
Brendan MacLean responded:  2020-04-26

Hi Anatoly,
I expect that Skyline is handling this as we would expect, give the RegEx already in place to extract this value:

   private static readonly Regex REGEX_IRT = new Regex(@" iRT=([^ ,]+)");

However, Skyline is still likely waiting for you to define which peptides are your standards and what standard iRT values you want to use. I am sure we could make this better, but I think all you need to do is define a new iRT library in the Peptide Settings - Prediction tab (click the button with the calculator on it), choose your standards from the available list and then click the Add button and choose "Add Spectral Library". Then point Skyline to the library for your .msp file. This should allow Skyline to import and calibrate all of your iRT values to the standard.

I am sure we could improve this workflow to auto-detect the standard in your library from our existing list, as we have been doing in other cases recently. But, for now, this extra step is required. Hopefully, you find it easier than your conversion script.

Thanks for sharing your experience with a downloaded Prosit .msp file. Have you tried building your Prosit libraries directly in Skyline? That should really be the most straightforward way of achieving your desired end-result.

--Brendan

 
anatoly.urisman responded:  2020-04-26

Hi Brendan,
Thanks for the quick reply.
I am using Skyline-daily v20.1.1.83, which I think is the newest version.
I have not been able to add a Prosit .msp library in any way other than using Settings --> Peptide Settings --> Library --> Edit List --> Add...
And this operation does not let you specify an iRT database. Please note that my document already has iRT peptides added as targets and the iRT calculator is set accordingly. Upon the addition of this Prosit library (which contains spectra for the iRT peptides), I do not see iRT values for any spectra when I browse the library in View --> Spectral Library Explorer. The values are visible if I use my manual work-around trick by adding "RetentionTime: ###" line in the .msp file.

Unfortunately, I can't get .msp into Skyline any other way. I tried adding it as a library to an existing iRT calculator as you suggested above, and get an error that says ".msp is not a BiblioSpec or Chromatogram library. Only BiblioSpec and Chromatogram libraries contain enough..."
I also tied to build a new library but get an error that says that ".msp is not a supported file format". This was the case with both "Add peptide search" wizard and with Settings --> Peptide Settings --> Library --> Build...

To answer your question about Prosit library generation directly from Skyline -- yes, I've been able to do it. This is a fantastic addition to Skyline! It appears to work as intended (including iRT parsing). I also like that .blib is generated in the process. Some users may find it confusing that in order to specify peptides to be included in the library, they must be added to the document as targets. Personally, I like to separate target selection from library generation and would prefer a cvs input file similar to that on the Prosit website.
A couple of questions re this workflow:
Is there a way to try the new (unpublished) Prosit "_preview" model directly from Skyline?
Is there a way to generate a mirror plot to compare spectra for a given precursor from two different libraries (e.g. Prosit vs experimental)?

Thank you to the whole team for the outstanding work on Skyline!
Anatoly

 
Brendan MacLean responded:  2020-04-27

Hi Anatoly,
That is the right way to add the library. Now you just need to go to Peptide Setting - Prediction, click the calculator button and choose Add. Once in the Edit iRT Calculator form, click Create and locate the new .irtdb file you are creating. Then you need to choose your desired standards from the available list, or create your own. Then you need to click the Add button at the bottom and choose Add From Library. Next choose the library you added for you .msp file. Skyline will import all of the iRT values from that library into this separate iRT library in the .irtdb file. Only .blib files support having the IRTDB inside the .blib file. This is because, although, the .msp file contains iRT values, there is nothing specifying which values are the landmark peptides used to calibrate everything else.

Hope this helps get you past your current blocking issue. Keep trying. It really is possible and even pretty easy once you know what to do. Sorry it is not more intuitive.

--Brendan

 
anatoly.urisman responded:  2020-04-27

Hi Brendan,
Yes, this works!
However, to get back to my original question/comment, the iRT values from the library still do not dispay in View --> Spectral Library Explorer
Not a huge problem - perhaps something to note for a future version.
Thank you again for your help.
Anatoly