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Automatic small molecule transition extraction from heavy + light neat runs

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Automatic small molecule transition extraction from heavy + light neat runs skyfall  2024-12-15 06:34
 

I am running heavy standards with the corresponding non-labeled ones neat in PRM mode. I have added all precursor adducts in Skyline. If I don't know the transitions or want all the transitions that are optimal in my set-up, is it possible to automatically extract them based on the mass shift between heavy and light?
Or do I need to buy small molecule spectral libraries?

(coming from proteomics, so sorry for my missing knowledge)

Thanks a lot!
 
 
Brian Pratt responded:  2024-12-15 10:19

As you probably know, Skyline can readily predict fragment ions for peptides. But fragment prediction for generalized small molecules is beyond Skyline's capability. Spectral libraries are one way to go - and there are several free options there - and there do exist tools such as LipidCreator (also free!) that can do this for specific molecule types.

Thanks for using the Skyline support board,

Brian Pratt
 
skyfall responded:  2024-12-15 11:26

Thanks for your response!
So there is no way to get fragment traces for heavy light pairs that have a fixed mass shift (like the fragments that include the label position)? They are easy to spot in the MS2 spectra, but hard to extract in batch. Based on their coelution (heavy light) they provide very strong evidence. I wonder how transitions are defined in the first place before they become part of a spectral library. Is there a way for me to search my neat runs or generate a spectral library from them?
 
Nick Shulman responded:  2024-12-15 11:42
Skyline cannot infer the heavy transitions from the light transitions because Skyline does not know, in general, how many of the heavy atoms any particular fragment will contain.
But if you know what the m/z's of the heavy transitions should be you could add them to your Skyline document by right-clicking on the precursor in the Targets tree and choosing "Add Transition" or adding a list of transitions with the "Edit > Insert > Transition List" menu item.

After you have extracted chromatograms for your data you can create a spectral library using the "File > Export > Spectral Library" menu item.
That will create a .blib file with pseudo spectra in it where the m/z's in the spectra are the m/z's of the transitions in the Skyline document and the intensities are the integrated chromatogram peak areas.

-- Nick
 
Brian Pratt responded:  2024-12-15 11:42

Skyline's not really a search tool - it's for targeted mass spec. You can certainly manually create a text-based transition list based on what you see in the raw data, but you're probably better off using a proper search tool.

I don't have any recommendations for that (other than suggesting LipidCreator if you're doing lipids) but perhaps someone else monitoring this list has suggestions. It would be helpful to know what kind of molecules you're studying.
 
skyfall responded:  2024-12-17 04:31

Thanks! Predictions for vitamins or steroids would be interesting. 1-2 transitions are known, but with PRM comparing heavy with light runs, there are way more nice patterns that shifted by the label mass only. I guess I am just used to several transitions from proteomics and was hoping for sth. similar :)