Skyline for fully labelled metabolomics

support
Skyline for fully labelled metabolomics adelabriere  2020-02-10 02:50
 

Dear Skyline team,

We are trying to use skyline for fully lablled cell extract for lipidomics analysis, with the isotopic pattern going up to +20 in some case.

Is there a good setup to do so ? It seems at the moment that skyline does not allow us to go above 10 peaks and has a threshold which prevent the detection of heavier isotopes.

Best regards,

Alexis Delabriere

 
 
Nick Shulman responded:  2020-02-10 11:41
It sounds like you are using Skyline for small molecules.

Skyline requires you to specify which atoms are heavy labeled and also what the atom percent enrichment of that labeling was.
Skyline uses the following symbols to indicate heavy isotopes:
2H: H'
13C: C'
15N: N'
18O: O'
(there are a few other symbols available).

If you just want Skyline to look for the heavy version of a molecule, you can use those symbols in the chemical formula when you do:
Edit > Insert > Transition List (small molecules)
If you want to specify light and heavy pairs of precursors then you have to specify the heavy isotopes in the "adduct" column of the Insert Transition List form. I am not an expert on how to specify adducts in Skyline, but if you need it, we can probably point you at some pages that describe how to do it.

For instance, if you wanted to add D3 leucine to your Skyline document, you would specify that its chemical formula was:
C6H10NO2H'3

If your isotope labeling was not 100% effective, then you can specify the atom percent enrichment at:
Settings > Transition Settings > Full Scan MS1 filtering > Isotope labeling enrichment

So, to answer your question, you are always telling Skyline to look for a particular heavy or light form of a molecule. You cannot tell Skyline to look for all forms of the molecule with an amount of 15N enrichment ranging from 0% to 100%. That sort of labeling would result in an isotope pattern that was potentially smeared across dozens of Daltons.
We never figured out how to model that sort of labeling, and therefore it really is not possible to analyze that sort of data in Skyline.

So, if you are doing an experiment where you were feeding an organism a heavy labeled diet of 15N amino acids over a short period of time, and you are not sure how much of that 15N got incorporated into the proteins, and how much 14N is still present, then you will probably not be able to analyze your data in Skyline.
But, if you are doing a SILAC experiment where you know the level of enrichment inside of the cells matches the media that they were grown in, then it should be possible to make this work in Skyline.

Hope this helps,
-- Nick