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Small molecule isotopic peak SChen  2025-11-12 10:54
 

Dear Skyline team,

I am using the small molecule interface in Skyline 25.1.0.237. I am trying to see the top three isotopic peaks (M, M+1, M+2). I changed the settings according to the Adding Isotopic Peaks section in the Hi-Res Metabolomics tutorial.

  • Transition Settings - Full Scan: Peaks is set to 3, Retention time filtering set to "Include all matching scans".
  • Transition settings - Filter: Precursor adducts: [M+H], ion types: p. Auto-select all matching transitions is checked.

I am still not seeing the isotopic peaks for all molecules (some molecules have all three isotopic peaks, some only have one or two). Any suggestion is appreciated!

Thank you,
Shimin
 
 
Brian Pratt responded:  2025-11-12 11:52

When you say "not seeing the isotopic peaks", do you mean that they do not appear in the Targets tree, or do you mean that Skyline isn't finding them in the mass spec data?

Thanks for using the Skyline support board,

Brian Pratt
 
SChen responded:  2025-11-12 12:10

Hi Brian,

Thanks for the response. I mean the M, M+1, and M+2 ions do not appear in the Target tree.

Shimin
 
Brian Pratt responded:  2025-11-12 12:25

I think it's probably because Skyline doesn't think your mass spec can resolve those particular isotopes, based on what you've told it about resolution.
 
SChen responded:  2025-11-12 13:14

Brian,

I realized that all carbon and nitrogen in my molecules were isotopically labeled (13C and 15N), which is probably the reason why I don't see isotopic peaks in the Target tree. S in Cystine is not isotopically labeled and is the only one that has M, M+1, and M+2 peaks in the Target tree. My question is resolved. Thank you for your help.

Best,
Shimin
 
SChen responded:  2025-11-19 14:34

Hi Brian,

On a similar topic, is there a way to add [M-1] precursor isotopic peak to the Target tree? I tried modifying Transition Setting - Full-Scan - Peaks: 5. It does not seem to bring up the [M-1] precursor isotopic peak.

Thanks,
Shimin
 
Mike MacCoss responded:  2025-11-19 20:55

Hi Shamin,
The easiest way to do that is to right click the precursor to bring up the attached menu item and select "pick children".
Precursor Right Click

Then you click the funnel and you should see the [M-1] isotope as an option.
Precursor Funnel Click

Precursor M-1

If your data is already imported you will need to reimport the data but that should go pretty quickly.

Hope this helps,
Mike
 
SChen responded:  2025-11-20 06:43

Hi Mike,

Thank you for the suggestion. This works!

Best regards,
Shimin