Peak extraction window shifting

support
Peak extraction window shifting rmolden  2020-06-18
 

Hi,
My retention times shift very little from run to run, so I would like to use a stringent retention time filtering (0.5 minutes of MS/MS IDs) when chromatograms are extracted to reduce noise. When I import my results I notice that for some peptides the window in which XIC are extracted shift from run to run (see attached), even though the library is built just from one run and I have not applied any iRT calibration. Is there any way to make sure that that extraction window stays fixed across runs?
Thanks!
Rosie

 
 
Nick Shulman responded:  2020-06-18
Rosie,
Can you send us your Skyline document? I will try to figure out how Skyline came up with the times that it chose.

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

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

When Skyline is deciding what retention time to use for a particular peptide in a particular replicate, Skyline does something a little different if the replicate's name matches the name of one of the source files in your spectral library. However, if you're saying that your library was built from only one run, I cannot think of a reason you would be seeing the behavior you are seeing.
-- Nick
 
rmolden responded:  2020-06-18
Thanks Nick,

Unfortunately I can't share the skyline file with you. I double checked my library and I had added one of the other files in (the one with the shift). I made a copy of the file and changed the name and reimported and it got rid of the shift in the XIC window that I had seen.

If I want to create a library from more than one file, and don't want to change their names so I can still see where the MS/MS IDs are is there any way to do that without the shift in the XIC window? In the example I sent, most of the MS/MS IDs only come from one of the files in the library and the actual shift in retention time from run to run is much smaller than the shift in XIC window that Skyline is applying.

Best,
Rosie
 
Nick Shulman responded:  2020-06-18
You might get some useful information if you use the menu item:
View > Retention Times > Alignment
This brings up a window where Skyline shows the times of the MS/MS IDs from your spectral libraries, and shows what slope and intercept Skyline would use to map those times onto a different file.

.blib files are SQLite databases. If you are familiar with database tools, you can read the list of peptides, retention times and files by JOINing the RefSpectra, RetentionTimes and SpectrumSourceFiles tables.

Hope this helps,
-- Nick
 
rmolden responded:  2020-06-18
Yes it helps, thank you for the explanation!