RT issue when one peptide and it's own modified peptide co-exsit

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RT issue when one peptide and it's own modified peptide co-exsit qi tang  2018-05-15 07:32
 

Dear Skyline Group,

I have one peptide and it's own modified peptide. One RT is 68.21 and the another is 45.71(See attached picture 1.png and 2.png). The extraction window is set to 3 min(See attached picture 3.png). I found the actual extraction window is from 42.71 to 71.21. Why? Can I avoid it? I just want extract the peptide peaks as it's been set to(3 min window separately).

Best,
Qi

 
 
Brendan MacLean responded:  2018-05-15 09:07

Hi Qi,
I wouldn't expect these two to be related at all. Can you right-click in the graphs and click Peptide ID Times > Other Runs. That should show you light blue lines where the IDs for the peptides were found, which should explain why Skyline is extracting the range it is. I expect you will see that your search engine identified both variants at around 69 and 46.

What search method are you using to build your library? Seems strange that it would end up with matches to both peptide molecules at both times.

Thanks for posting the screenshots. Hope we can get to the bottom of what is going on.

--Brendan

 
qi tang responded:  2018-05-15 10:13

Hi Brendan,
Thank you for your quick response.

I use BlibBuild.exe to build my own specific library. I write RT information directly into .ssl file(see attached picture 6.png).

I did what your suggest(right-click in the graphs and click Peptide ID Times > Other Runs). Attached is the results. The light blue lines showed both around 69 and 46(see attached picture 5.png and 6.png).

Thanks again.

Best,
Qi

 
Brendan MacLean responded:  2018-05-15 10:30

Hi Qi,
Skyline really shouldn't be matching ID times to both modified peptide forms unless that is what your SSL contains. But, if you are sure that is not the case, then we probably need your SSL and supporting .ms2 files (zipped) and the resulting .blib library file.

You can post them to:

http://skyline.ms/files.url

If they all check out as expected, then I guess there must have been some kind of bug introduced into the Skyline peptide to spectral library matching algorithm. Though, that would seem like it would break a lot (if Skyline were just matching based on the unmodified sequence). So, it seems unlikely.

Well, let's start with your SSL files and spectral library to see what is in those.

Thanks for the screenshots.

--Brendan

 
Brendan MacLean responded:  2018-05-15 10:55

It occurred to me that in 6.png you only show the two peptides identified in a single file (_land4.ms2). Are there other .ms2 files represented in you SSL file, and is it possible that the retention times in those explain the two ID lines you are seeing for the two different peptide forms?

Please have a close look at your SSL and all entries corresponding to the two peptide forms, and let me know if you are completely confident that the unmodified form always appears around 68.2 and the modified form always appears around 45.7.

If so, then getting those files really is the next step.

--Brendan

 
qi tang responded:  2018-05-16 08:38

Hi Brendan,
I did all I can to look for my mistakes, I just can not find any in my knowledge. Please check it for me.

All peptides which co-exist unmodified form and modified form have this issue in all runs.

I can't upload my files into the address you gave to me. said "You do not have privileges to this directory. Verify that you are signed in appropriately". I already signed and used Chrome browse.

The zipped file is small(included .ssl, .ms2, .blib files), so I just attached.

If there anything else I can do, please let me know.

Thanks for your help.

Best,
Qi

 
Nick Shulman responded:  2018-05-16 14:31
Hi, Qi,

Your file was successfully uploaded. We have a problem with our website where it says "Permission denied" if you try to upload the same file more than once, instead of saying "File already exists".

I believe the problem that you are encountering is the same one as in this support request:
https://skyline.ms/announcements/home/support/thread.view?rowId=35707
Skyline had a bug where it did not pay attention to modifications when decided how long of a window to extract chromatograms over.

This bug has been fixed in the current Skyline-Daily. It has not yet been fixed in the released version of Skyline 4.1, but it will be fixed the next time we make an update of Skyline 4.1.
 
qi tang responded:  2018-05-17 08:02
Hi Nick,

Thank you for your help. I checked what you said. It's real the newest SkylineDaily already resolved my problem.

Thanks a lot.

Best,
Qi