Prosit in skyline Tobi  2019-12-05
 

Dear all,

thank you very much for integrating Prosit into Skyline. I noticed that the dotp in the new library match mirror view does not treat y ions from deferentially labeled peptides as equivalent. Is it possible to extract the Comparison dotp via results grid for all peptides at once?

Best,
tobi

 
 
Tobi responded:  2019-12-06

Edit:

Being able to quickly adapt the Prosit NCE in the Library mirror view is great, having this available for the dotp between Prosit library and measurement would be nice (for example under Tools/Options/Prosit when changing the NCE there I would expect to see a change in dotp for the data or in the target list.

Also the issue with isotope-labeled y-ions can be seen everywhere, meaning Prosit in Skyline can only be used for light peptides as of now.

In total this is still a powerful imporovement, thank you very much for all the great features being added, it means a lot.

Best,
tobi

 
Nick Shulman responded:  2019-12-06

Tobi,

Can you send me your Skyline document? I probably can reproduce this problem without that, but it will make things easier for me.

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms and spectral libraries.

If that .zip file is less than 50MB you can attach it to this support request.
Otherwise, you can upload it here:
https://skyline.ms/files.url

Thanks!
-- Nick

 
Tobi responded:  2019-12-07

Dear Nick,

thanks for the swift response and sry I forgot that, its quite small so see attached, hope it contains everything.

Best,
tobi

 
Nick Shulman responded:  2019-12-09
tobi,

Thank you for sending that file.

The problem is that when Skyline creates a spectral library from Prosit, Skyline uses the spectra for the light versions of the peptides, but writes the heavy version of the Peptide Modified Sequence to the .blib file. Therefore, when Skyline displays the spectrum to you in the Libary Match window, Skyline thinks that it's supposed to be the heavy version of the spectrum. For this reason, the masses of all of the y ions are incorrect by a few daltons, so none of the y ions get annotated properly.

I will fix this so that when you create a .blib file the spectra have the correct modified sequences.

I hope this will fix the problem that you reported. I am not sure that I understood everything that you have said here.
-- Nick
 
Tobi responded:  2019-12-09
Dear Nick,

thanks for the great response.

The dotp in the mirror view should consider heavy labels as well as different sets of ions ( a selfmade library might not contain y1 and y2 but prosit does and this should not reduce the dotp).

Thank you very much for the fix, its an exiting feature and quite important as it impacts retention time standards as well.


Best regards,
tobi
 
Brendan MacLean responded:  2019-12-09
That should definitely help. Yeah, that is a bug. It took some explaining (to Tobi Rohde who implemented the features) that we needed to communicate the structurally modified peptide to Prosit without the isotope labeling modifications (because Prosit doesn't understand those and they don't impact fragmentation) and that Skyline was already built to match spectra assigned to the light modified sequence to the heavy precursor inside of Skyline.

But, if the library contains a spectrum for the light modified sequence labeled with the heavy sequence, then, as Nick notes and you have seen, the translation won't work correctly.

Thanks for reporting the issue and providing files.

--Brendan