Issues with Skyline Chromatography (Metabolomics)

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Issues with Skyline Chromatography (Metabolomics) victoria roberts  2023-08-16 10:03
 

I am having issues with peaks showing up on skyline, it shows that they are there in other softwares like qual browser and freestyle but whenever I try to look at it through skyline it looks like there wasn't really anything showing up although there was other random peaks (so it wasn't an upload issue). I think it may have to do with mass tolerance but I tried changing the transition settings to see if I the peaks would show up but they haven't, even when I went to a match tolerance of 0.5 m/z, although they definitely show up on other softwares within 0.005 m/z. I am not sure if there is something hard-coded that is preventing me from seeing the peaks or if I just don't know how to use the settings correctly, but I would appreciate any help I can get.
Thanks!

 
 
Nick Shulman responded:  2023-08-16 10:10
Can you send us your Skyline document and your .raw file?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

If that .zip file and/or your .raw file are less than 50MB you can attach them to this support request. You can upload larger files here:
https://skyline.ms/files.url
-- Nick
 
victoria roberts responded:  2023-08-16 10:20
Hi Nick! I just uploaded it to the link that you provided.

thanks :)
 
Nick Shulman responded:  2023-08-16 10:36
Thank you for uploading that zip file.
Can you also send us the file "20230725_IntradaOA_Koley_90ACN_VR_TM_MC-TM-45_fullscan_neg_113_WT_con1.raw"?

I see that most of your chromatograms are zero most of the time and only have a non zero value at sporadic places.
When Skyline extracts chromatograms from spectra like this, Skyline sums up all of the intensities across a m/z window centered around your molecule's m/z.
The width of the channel that Skyline sums across is controlled by the "Resolving Power" setting at "Settings > Transition Settings > Full Scan".
I see that you have the resolving power set to 120,000. It might be that if you change that to a smaller number you will get better chromatograms.

After you change the resolving power you should tell Skyline to extract chromatograms again by pushing the "Reimport" button at "Edit > Manage Results".

By the way, if you want to see how wide of a channel Skyline is summing across, you can click on a point along the chromatogram. When you click on a point along the chromatogram, Skyline brings up a spectrum viewer which will show shaded regions around the places where Skyline summed intensities to extract chromatograms for particular transitions.

Also, for Thermo data, we usually suggest that you set the "Mass Analyzer" at "Settings > Transition Settings > Full Scan" to "Centroided" because Thermo's centroiding algorithm does such a good job of separating signals from things with neighboring m/z values. When the mass analyzer is "Centroided", the other setting becomes "Mass Accuracy" instead of "Resolving Power", but it still controls the width of the channel that Skyline sums across when extracting chromatograms.
-- Nick
 
victoria roberts responded:  2023-08-16 10:40
Thank you so much! I'll try this out
-Victoria