Quantifying on multiple fragment ion isotopic peaks

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Quantifying on multiple fragment ion isotopic peaks johan gobom  2020-07-08
 

Dear Skyline Team,

I’m setting up a PRM method for large peptides, around 4.5 kDa, +4-charged, on an Orbitrap. For many of the fragments, the monoisotopic peak is not the strongest by far, so I would like to quantify on up to four isotopic peaks for each fragment. Is this possible to do in Skyline?

All the best,

Johan

 
 
Brian Pratt responded:  2020-07-09

Hi Johan,

Yes, this is a fundamental capability of Skyline. You can set the isotopic peak count in the Transition Settings | Full Scan window.

Thanks for using the Skyline support board,

Brian Pratt

 
Brendan MacLean responded:  2020-07-09

Um, no. This is not possible. We have never heard this request before and at present Skyline always uses either the monoisotopic peak or the average peak m/z, which would only work if you were willing to make your extraction width wide enough to capture the signal in that region, which actually might work okay for PRM, but you would be sacrificing selectivity and making it more like SRM.

So, far, I think we have been getting away with mostly smaller fragment masses and +1 and +2 charge states.

The setting Brian mentioned is for precursors extracted from MS1 scans, not for fragments extracted from MS/MS scans as in the PRM you mentioned.

Sorry, we don't have an easy way of enabling what you are looking for.

--Brendan

 
Brian Pratt responded:  2020-07-09

Oops, yes - "fragment" was the key word there. Sorry if I got your hopes up!

Brian