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Two integration/importation issues

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Two integration/importation issues avollaro  2018-07-23 12:24
 

Hi there,

I am using Skyline 4.1.018169 for metabolomics data, and I had a couple issues.

First, while looking at acetylcholine, Skyline displayed a different retention time window for one sample "run 16" of 50. In addition, it looks like the .raw file imported transition 148->87 m/z, but did not import transition 148->60 m/z data for run 16. Please see the attached PowerPoint file "Skyline AC Issue.pptx". I included a view of the .raw chromatograms in MassLynx, which have the correct retention window and a peak for the run 16 148->60 m/z transition.

Second, Skyline frequently misses the integration of a peak for a single transition. In the attached example for run 37 of 50, the gamma-aminobutyrate 104->87 m/z transition is not integrated, but the peak is shaded as if integration was performed. The second transition 104->86 m/z for run 37 has been integrated successfully, and all prior/subsequent runs for this transition were integrated successfully. However, instances of missed integration happened for 9 separate metabolites in this data set It is frustrating to have to redo the integration of several peaks throughout large data sets. Is there any solution for this? I have already tried using "Apply Peak to All," but this reintegrates all the peaks and usually leads to more corrections of improper integration (many times Skyline does not capture the entire peak area).

Best Regards,
Alyssa Vollaro
 
 
Nick Shulman responded:  2018-07-23 12:44
In "Skyline Integration Issue.pptx" it looks like it might have something to do with the menu item:
Settings > Integrate All
When "Integrate All" is turned off, Skyline will not integrate transitions whose peaks are not exactly centered with the other transitions in the molecule. Skyline does this in order to make it easier to find transitions that might have interference.

I cannot think of anything that would be causing the behavior that you are seeing with "Skyline AC Issue.pptx".
Can you send us your files?
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

You should send us that .zip file as well as the raw file for "run 16".
(You are welcome to send us more files if you think they might help).

If each of those files is less than 50MB, you can attach them to this support request.
Otherwise, you can upload them here:
https://skyline.ms/files.url
 
avollaro responded:  2018-07-23 13:31
Hi Nick,

Thanks for the fast response! I turned on "Integrate All" and that did the trick.

I have attached the requested Skyline .zip and the .raw file for run 16. I appreciate you looking into it!
 
Nick Shulman responded:  2018-07-23 14:25
I would recommend that you upgrade to Skyline-Daily.

Your Acetylcholine has a precursor mz of 146.02, and product mzs 87 & 60.
In your .raw file, there are two chromatogram groups: 146.01/{86,87} and 146.02/{87,60}.

The correct behavior would be for Skyline to use the "146.02/{87,60}" chromatogram group from your .raw file. Unfortunately, Skyline 4.1 has a bug in it where, if you have specified an explicit retention time, then Skyline will prefer chromatogram groups whose largest peak is closer to the explicit retention time.
This behavior is not good, and can lead to very confusing behavior where one of your replicates is matched to a different set of chromatograms. In Skyline 4.1, for replicate 16, Skyline ends up deciding that the 146.01/{87} chromatogram is a better match than 146.02/{87,60} pair of chromatograms.

If you upgrade to Skyline-Daily, then Skyline will always match that Acetylcholine to 146.02/{87,60}.
When you first open this in Skyline-Daily, I would recommend that you do:
Edit > Manage Results > Rescore
so that Skyline will go through and recalculate all of the peak areas, in case any of the wrong chromatograms were previously being used.