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MRM with known peptides c frampas  2025-01-27 09:32
 

Hello,

I am trying to optimise my MRM method and wanted to use Skyline. I am looking at 3 synthetic peptides, each heavy labeled. In Skyline, I tried to add the fasta with my 3 peptides but it is giving me this chat: This operation has added 3 new proteins with no peptides meeting your current filter criteria. I have also tried to run a peptide seach with no success. I have 3 mzml files corresponding to my PRM runs.

Could I have some info as to how to proceed so I can look at my data?

In case that helps, my fasta with my 3 peptides looks like this:

CRP| DYSLFSYATK^
DYSLFSYATK

Is it actually possible to add information on the fasta to show it is heavily labeled on the C-terminus?

Any help would be greatly appreciated.

Best wishes,
Cecile
 
 
Nick Shulman responded:  2025-01-27 09:41
A different way to get these peptides into your document would be to use the menu item:
Edit > Insert > Peptides

When these peptides are inserted into the document the will just be in a generic Peptide List, but then you can reorganize the peptides and add protein information using the "Refine > Associate Proteins" menu item and pointing Skyline at your FASTA file.

After the peptides are in your document you can right-click on them in the Targets tree and choose the "Modify" menu item.

The following Webinar has a lot of good information about working with modifications in Skyline:
https://skyline.ms/Webinar10.url
-- Nick
 
Mike MacCoss responded:  2025-01-27 09:43

Hi Cecile,
I would suggest looking at the Tutorial on Existing Quantitative Experiments.
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_existing_quant&show=html

It illustrates how to build a Skyline document with heavy labeled peptides and how to import data that was collected without using Skyline to create the method (which is how we recommend).

Best of luck with your analysis,
Mike
 
Mike MacCoss responded:  2025-01-27 09:44

There is also this great webinar on the use of isotope labeled standards in Skyline which builds on the webinar Nick suggested.
https://skyline.ms/project/home/software/Skyline/events/2015 Webinars/Webinar 12/begin.view?
 
c frampas responded:  2025-01-28 07:16

Hi both,

Thank you very much for your help!

Best wishes,
Cecile