Allow integration of multiple isotopes for high molecular weight fragments (Top-Down PRM)

Allow integration of multiple isotopes for high molecular weight fragments (Top-Down PRM) v delcourt  2018-10-10


With recent analytical chemistry publication, there's something interesting to do with multiplexed top down PRM (

While doing some experiments on my side, I thought this was indeed interesting, but there might be something to add to Skyline to complete data analysis of these experiments. Indeed, in case of high molecular weight (with charge > 1), it's very likely that fragments' most abundant isotope would not be the monoisotopic. However, Skyline seems to integrate only the first one (see capture attached). Maybe this feature is already implemented but I couldn't find it.

By adding all/multiple isotopes to target ions integration, this would increase detected signal intensity which would affect results significantly.

I can send data if needed.
Best regards,

Nick Shulman responded:  2018-10-10
There is currently no way to do this in Skyline.

I actually made a poster about what it would look like if Skyline were extracting the full MS2 fragment isotope envelope from DIA experiments. I was able to find one peptide that looked really good, but for most peptides, I think, looking at more m/z values in the MS2 increased the chance that you will have interference.

With a PRM experiment, the isolation window is usually (I think) .7 units wide. The precursor has to be at least charge 3 in order for anything other than the monoisotopic precursor to have been isolated. The monoisotopic precursor only produces monoisotopic fragments. The M+1 precursor produces a mixture of monoisotopic and M+1 fragments. One interesting feature of extracting the fragment isotope envelope would be for Skyline to report the fragment isotope dot product, but in order to do that, Skyline would need to know exactly how sharp the edges of the precursor isolation window were.

I was not able to find a scenario where this would actually help, but it would be great to take a look at your data.

You can upload your files here:

If you are sending us your Skyline document, you should use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files, including extracted chromatograms.
-- Nick
v delcourt responded:  2018-10-10

Thanks for your quick answer. In facts, this is true for peptides, but for large peptides or full length proteins, this feature would I believe be interesting.

Are joined two files with 1 large peptide (> 3 kDa) and one full length protein (21 kDa) with both acquired in multiplexed PRM as shown in publication I mentioned.

Also joined : Xcalibur integration of single isotope of first post mentioned fragment (top) and integration of same fragment but for 5 isotopes (bottom).

Best regards,

EDIT : Sorry, I noticed the procedure for creating the zip file after upload
v delcourt responded:  2018-11-02

Is this feature still in discussion on your side ?

Best regards,
Martijn van Duijn responded:  2018-12-11
Hi, I was also trying to integrate top-down PRM data of an intact protein into Skyline. My protein chain of interest is about 23 kDa, multiply charged in ESI. In order to circumvent the isotope problem, I wanted to try to use xtract (xcalibur on orbitrap data) to deconvolute all my MSMS scans of my precursor of interest, e.g. the 16+ of my protein. In the decharged/deisotoped MSMS spectra only a single peak remains for each fragment ion, which should be ideal for skyline. I ran into the problem of the maximum m/z limit that is allowed in Skyline. As detailed in this support ticket Issue 533 the limit is now somewhat arbitrarily set at 10,000. It would be helpful, to me at least, if this could be raised further for that purpose.