Chromatogram information unavailable in the Decoy

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Chromatogram information unavailable in the Decoy claire wu  2015-01-12
 
Hi,
I would like to get the FDR by using mProphet. I open the original .sky file and generate the decoy. But after re-import the .wiff file, it shows the "Chromatogram information unavailable" in the Decoy part. I remove all the .wiff and import the .wiff again, only the Chromatogram in the target part can be shown. In this way, I can't get the FDR. Could you give me some suggestions? Thanks in advance.

Claire
 
 
Nick Shulman responded:  2015-01-12
Your question sounds similar to this one:
https://skyline.gs.washington.edu/labkey/announcements/home/support/thread.view?rowId=10480

Is it possible that you pressed the "rescore" button instead of the "reimport"?

Reimporting should work. I will email you about how to send us files.
 
claire wu responded:  2015-01-12
Dear Nick,
Thanks for the prompt reply. I have tried the "rescore" but it still dose not work. Please let me know if you have further suggestions for this. Thanks so much.

Claire
 
Nick Shulman responded:  2015-01-13
Thanks for sending me your data files.
You have an SRM experiment, which means that the machine only collected for the masses that were specified ahead of time.

I believe that in order to use decoys in an SRM experiment, the decoy peptides need to be added to the instrument method before the data is collected.

What you are seeing is that there really are no chromotograms in the data file for those decoy peptides.
 
claire wu responded:  2015-01-13
Dear Nick,
Thanks so much for spending time checking my data. Is there other way that I can calculate the FDR since now the big data has been generated without adding the decoy peptides?

Best,
Claire
 
Brendan MacLean responded:  2015-01-13
Hi Claire,
You can use second-best peaks. Are you consulting the Advanced Peak Picking Models tutorial?

https://skyline.gs.washington.edu/labkey/wiki/home/software/Skyline/page.view?name=tutorial_peak_picking

It covers using second-best peaks on pages 15 and 26/27:

https://skyline.gs.washington.edu/labkey/_webdav/home/software/Skyline/%40files/tutorials/PeakPicking_2-5.pdf#page=15
https://skyline.gs.washington.edu/labkey/_webdav/home/software/Skyline/%40files/tutorials/PeakPicking_2-5.pdf#page=26

This is definitely your best resource for using the mProphet support in Skyline.

Thanks for using Skyline in your research.

--Brendan
 
claire wu responded:  2015-01-13
Hi, Bredan
I just found the tutorial and decided to use second-best peaks. It's good to receive the confirmation from you. Thanks so much for all the help and support from you and Nick.

Best
Claire