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[M+5] isotope peaks for small C-13 labelled molecules

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[M+5] isotope peaks for small C-13 labelled molecules CM  2025-06-04 04:10
 

Hello Skyline Team,

I was reading this discussion thread hoping to find some answers for my current situation and am wondering if anything has changed with regards to the MS1 filtering feature since the original post in 2019. I am working with MS1 data from small molecules labelled with C-13 and am hoping to see isotope peaks up to M+5 in some cases. However, I can only see up to M+3. I currently have the Full Scan settings set to Isotope Peaks included = Count, and Peaks = 6. Would altering the "Isotope labelling enrichment" settings under the Full-Scan transition settings tab help with this or is there another option to see those higher M+X isotope peaks?

I am working on a Windows 11 device and am currently using Skyline 24.1.

Thanks so much for your helpful software and support.

CM
 
 
Nick Shulman responded:  2025-06-04 04:21
Skyline still will only allow you to choose masses whose predicted abundance is at least 1% of the most abundant mass.
If you had a larger molecule then you would be able to add the M+5 Transition, but, if Skyline is not allowing you to add that M+5 then it means that Skyline does not think that mass has a high enough predicted abundance for you to want to extract a chromatogram for it.

Is there a particular molecule were you hoping to extract the M+5 chromatogram for? Is there a reason that you are interested in that mass, even though its predicted abundance is so small?
-- Nick
 
Nick Shulman responded:  2025-06-04 04:29
By the way, if you have artificially labeled the molecule yourself, then you should tell Skyline that the chemical formula of the Precursor is different.
The symbol that Skyline uses to represent 13C is:
C'
Usually, for small molecules, heavy labeling is specified in the adduct, in which case the chemical formula for five 12C being replaced by 13C would be:
C'5-C5
You can read more about small molecule adducts in Skyline here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=adduct_descriptions

-- Nick
 
CM responded:  2025-06-04 08:46

Thank you for the quick response!

I am doing metabolic flux analysis and would like to see those higher M+X (up to the number of carbons in each molecule) peaks for the isotopomer mass distribution vector. Mostly TCA cycle compounds. The data was collected by a targeted SIM method on a QE Plus with the isolation window offset to capture up to +7 m/z.

Re: your second response, so far I have been inputting my transition list (attached, QE isotopic labelling.csv) using compound name, formula, adduct, and charge, for the unlabelled compounds, then specifying the MS1 filtering to pick up the isotope peaks. I was mainly following this page. I just found this thread and it sounds like a very similar case - so I wonder if I have been inputting my transition list incorrectly? I did attempt modifying my list to have an individual transition for each precursor as you described (transition list attached, QE isotopic labelling 250604.csv), but that did not seem to work as it then only picked up on some isotopomers but not even the unlabelled peak.

CM