Nick Shulman responded: |
2024-07-25 09:34 |
On the Full Scan tab of the Transition Settings dialog you should change "Isolation Scheme" to something else such as "Results only".
You currently have that set to "All Ions" which tells Skyline that it should ignore what the mass spectrometer says was isolated in the MS2 spectrum, and just assume that product ions from all precursors can be found in the spectrum.
All MS2 spectra will contribute to the extracted chromatogram for all of the precursors in your Skyline document.
Your chromatograms look jagged because they are made up of some MS2 spectra that did isolate your precursor of interest, and some spectra which isolated something completely different and therefore have very little signal for the measured product ions.
If you change the Isolation Scheme to "Results only", MS2 spectra will only contribute to the chromatograms of the precursors whose m/z falls within the isolation window reported by the mass spectrometer.
-- Nick |
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Brian Pratt responded: |
2024-07-25 09:35 |
Hi Satoshi,
Have you examined the raw data in something like the SeeMS program to see if there is actually signal at the low points of the jaggies?
Thanks for using the Skyline support board,
Brian Pratt
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skimura responded: |
2024-07-25 09:56 |
Thank you both for the responses.
I changed the setting, but it did not change the chromatogram.
I have not examined the raw data, but it looks normal chromatogram in SCIEX software.
Satoshi
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Nick Shulman responded: |
2024-07-25 10:05 |
After you change that setting you need to tell Skyline to extract chromatograms again for that change to have an effect.
You can do that by using the "Reimport" button at "Edit > Manage Results"
--Nick |
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skimura responded: |
2024-07-25 10:16 |
I closed and opened a new window with a new setting, but it did not change.
I also did reimport, but it showed the same jaggy chromatograms.
Satoshi |
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Nick Shulman responded: |
2024-07-25 10:27 |
Can you send us your Skyline document and your .wiff file and the .wiff.scan file that goes with it?
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.
The Share Document dialog gives you the option to include the results files which will put the .wiff and .wiff.scan files into the .zip.
Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick |
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skimura responded: |
2024-07-25 10:45 |
Yes, sure.
Here is the file.
Satoshi |
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Nick Shulman responded: |
2024-07-25 11:12 |
If you click on a point along the chromatogram, Skyline will show you the spectrum that contributed to that point and you can push the wrench button to see the spectrum properties.
It appears that you have two sorts of spectra in your .wiff file. Some spectra have a collision energy of 20 and they seem to have a lot of signal for that product ion, and some spectra have a collision energy of 50 and those have no signal.
If you want to tell Skyline that it should only use a subset of the available spectra when extracting chromatograms, you can right-click on one of the precursors in the Targets tree and choose "Edit Spectrum Filter" and set things the way I have in the attached screenshot "EditSpectrumFilter.png".
After you have specified a spectrum filter you will have to reimport again with the Manage Results dialog.
There is some more information about Spectrum Filters on this page:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=SpectrumFilters
-- Nick |
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skimura responded: |
2024-07-25 11:43 |
It worked.
Thank you very much, Nick!
Can I ask the last question?
I tried to filter out spectra without signals by putting CE values in transition list as an attached picture, but it did not work.
Why does it not work?
Satoshi |
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Nick Shulman responded: |
2024-07-25 11:59 |
The "Explicit Collision Energy" value tells Skyline what collision energy to use when you export an SRM method.
It has no impact on chromatograms that Skyline extracts from spectra.
Using the explicit collision energy when extracting chromatograms is an interesting idea, but it is not something that we ever thought of.
-- Nick |
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skimura responded: |
2024-07-25 12:11 |
I understand it.
Thank you again. Your support was really helpful. |
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