How does Skyline treat shared fragment ions between precursors of same peptide from MSE data?

How does Skyline treat shared fragment ions between precursors of same peptide from MSE data? Juan C. Rojas E.  2021-07-07

Hi Skyline team,

I would like to confirm how skyline treats a fragment ion that is produced from multiple precursor ions of the same peptide when the data is acquired with MSE. Without the use of IMS it is not possible to discern from which precursor the fragment ion is being produced due to the lack of quadrupole isolation.

When the integrated fragment area is summarized for the peptide and a transition for the same fragment exists in two (or more) precursors, is the area of this "repeated" transition summed twice (or more) or does Skyline recognize it is a repeated value and only counts it once?

As always, thank you for your time and help.
Juan C.

Nick Shulman responded:  2021-07-14
Yes, those transition peak areas will be double counted by Skyline.

There are many cases where different transitions in the Targets tree are actually measuring the same signal, and currently Skyline always adds those areas to the Total Area of the Precursor that the Transition belongs to.

In the future, we hope to add features to Skyline to sometimes deconvolute those signals, particularly in the case where the MS1 signal of the isotope distributions of different precursors overlap.

In theory this same sort of deconvolution could be applied to the MSe signal of different charge states, but that would be much harder to do since there would need to be some way to predict the fragment proportions from each charge state.

-- Nick
Juan C. Rojas E. responded:  2021-07-20
Thank you Nick for the detailed and clear response.

I can see how that deconvolution for MSe be very challenging. Specially when variabilty introduced by different instrument settings can affect the fragment ion detected proportions. In the meantime I will write a script to stop counting "repeated" signals with the output of custom report.