Using Prosit for heavy ions

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Using Prosit for heavy ions denisoleynik3007  2020-05-08
 

Does Prosit work for a prediction of MS/MS spectra of heavy ions (in particular heavy y-ions)?

 
 
Brendan MacLean responded:  2020-05-10

We believe the Skyline-Prosit integration has been thoroughly tested with isotope-labeled peptide targets, including some initial bug fixes in this area. If you find any new issues with our implementation, please let us know.

 
denisoleynik3007 responded:  2020-05-10

Dear Dr. MacLean,
Thanks for Your rensponse. I've manually changed an already integrated peptide from DDA-based spectral library into a heavy one (by adding 13C(6) and 15N(2) to the C-terminal lysine), ran a Prosit algorithm and compared a manually changed heavy peptide and identical unchaged one. For the heavy peptide I got a dotp of 0.2122 (all y ions were unpredicted) and for the "light" unchanged one I got a dotp of 0.7661. Am I using an algorithm wrong? Initially, I wanted to spike into my sample some AQUA peptides and validate a Prosit on them but I wasn't sure if Prosit can predict a MS/MS spectra for heavy ions.

 
Brendan MacLean responded:  2020-05-10

Please provide clearer steps describing what you tried and what you are seeing, including screenshots. Also, what version of Skyline you are using. As I mentioned there have been fixes to the support for Prosit spectra for heavy peptides. It would also be good for you to turn on mirrored spectra in the Library Match view and send a screenshot of what you see. You can also attach a Skyline document (File > Share to create a .sky.zip file) to this request, or if it is over 50 MB, you can post it to our file drop point:

http://skyline.ms/files.url

Thanks for trying new things.

--Brendan

 
denisoleynik3007 responded:  2020-05-10

Dear Dr. MacLean,
There is what I tried to do:

  1. I have created a DDA-based library from the Skyline webinar #18 data set (for a sky.zip file please see a file drop point)
  2. Manually created a heavy ion (Right click on the peptide > Modify > Isotope heavy >13C(6) and 15N(2) to the C-terminal K of the peptide) from a random peptide.
  3. Launched a Prosit search (Tools > Options > Prosit)
  4. Mirror plot of a library match for a "light version" is shown in "Screenshot_1" photo
  5. Mirror plot of a library match for a "heavy version" is in "Screenshot_2" photo
    I would be grateful for any comments on how I can improve this. Thank You
 
denisoleynik3007 responded:  2020-05-10

P.S. I am using a Skyline version 20.1

 
Brendan MacLean responded:  2020-05-10

I am pretty sure I fixed that issue. It was reported in March:

https://skyline.ms/announcements/home/support/thread.view?rowId=43423

The fix has been released in Skyline-daily:

https://skyline.ms/announcements/home/software/Skyline/daily/thread.view?rowId=44289

But not yet in a 20.1 patch release. The fix has been committed to our 20.1 branch and will be in the next patch release:

https://github.com/ProteoWizard/pwiz/pull/989

Thanks for taking the time to post your feedback and clarify with screenshots.

--Brendan

 
denisoleynik3007 responded:  2020-05-10

Thank You very much for Your time and for clarification!