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Precursor metabolite data from SWATH

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Precursor metabolite data from SWATH lefelit  2020-04-09 09:02
 

Hi

I have metabolite data from TOF MS acquisition on a SCIEX triple TOF instrument and I am trying to extract the ion chromatogram information for more than 200 precursors. I am not interested at this stage for fragment data. I built a method with all the molecules of interest with the accurate masses and when i applied it to a test mixture of pure standards the software does not find almost 60% of them. I set the mass window to 0.2 and the RT window to 1min and still does not find them. What could be the source of the problem? How could I also customise the threshold setting to different value per precursor?

Many thanks
 
 
Nick Shulman responded:  2020-04-09 17:37
Do you want to send us your data files? We can take a look.

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

You should also send us one of your .wiff files and the .wiff.scan that goes with it.

If those files are less than 50MB you can attach them to this support request.
Otherwise, you can upload them here:
https://skyline.ms/files.url

-- Nick
 
lefelit responded:  2020-04-29 10:33
Hi Nick,

I uploaded a test mix injection (OROBA_T3) file to the drive to help you work through the data.

Below I attach the zip file with the method

Looking forward to hear from you soon
Filippos
 
Nick Shulman responded:  2020-04-29 12:45
Filippos,
Thanks for sending me your Skyline document. Are you also going to send a .wiff file and a .wiff.scan?

Is there a particular molecule and in a particular that you have a question about?

One thing that I see in your Skyline document is that many of your molecules have no precursors under them (for example "aKet"). Skyline will not extract chromatograms for a molecule unless it has a precursor listed under it. You can add a precursor to a molecule by right-clicking on the molecule in the Targets tree and choosing "Add Precursor".

It looks like your settings might have been different when you extracted chromatograms from your "1L" replicate. After you make a change to your settings, or after you add precursors or transitions to your Skyline document, you should tell Skyline to extract chromatograms again. You can do that by going to:
Edit > Manage Results > Reimport

It might be a good idea for you to take a look at some of our small molecule Skyline tutorials. You can find them on this page:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials

-- Nick