MS/MS data analysis related question

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MS/MS data analysis related question PP  2024-07-10 08:57
 

Hello,

I have acquired Full Scan MS2 data from Orbitrap Exploris 240. I wanted to know if Skyline is able to identify product ions based. I see green dots next to the precursors ion indicating that the precursors are present however all b,y,a ions are red. Is Skyline able to detect data only in MRM format or all MS/MS data?

 
 
Nick Shulman responded:  2024-07-10 10:02
Whether the symbol next to a transition is red or green depends on whether "Settings > Integrate All" is checked.
If "Integrate All" is checked, the symbol will be green unless the peak area is zero.
If "Integrate All" is unchecked, Skyline checks a few more things such as that the apex of the peak is within one quarter peak width of the apexes of the other transitions.

There is some more information about those colors on page 3 of the Method Refinement tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_method_refine

If you have more questions about your dataset it might be helpful if you send us your Skyline document.

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 also gives you the option to include your .raw files.

If that .zip file is less than 50MB you can attach it to this support request. Larger files can always be uploaded here:
https://skyline.ms/files.url

-- Nick
 
PP responded:  2024-07-10 10:17
Thank you for your response. I see green dots next to some of my product ions now. However, to see the peaks of those product ions, i am having to do Right Click->Transitions->Products. If I do Right Click->Transitions->All, it is only showing precursor. Shouldn't it be showing both precursor and product when selected All?
 
Nick Shulman responded:  2024-07-10 10:30
Yes, it should show both precursor and product chromatograms if you choose "Transitions > All".
One thing that might be happening is that the MS1 precursor chromatogram intensities are much higher than the MS2 product intensities and so the product chromatograms just look like a tiny bump above the X-axis.

The menu item "Transitions > Split Graph" will plot the precursor and product chromatograms on different charts with different Y-axes which makes it easier to see the product chromatograms if they are being dwarfed by the precursor chromatograms.

If you send us your Skyline document I might be able to give you a better answer about what is going on.
-- Nick