M+X peaks for fragment ions

M+X peaks for fragment ions sasha  2013-05-22 09:16
Dear Brendan,
Is there a way that i can see, or quantify, the isotopic clusters for fragment ions scanned at high resolution?
Brendan MacLean responded:  2013-05-22 11:16
Hi Sasha,
I knew that request would come eventually, but, unfortunately, I have not written any code to support this directly. It will make things more complicated for techniques like DIA/SWATH, since to have predictable M+ peaks for fragment ions, you must include the entire precursor isotope envelope in your isolation. Even for targeted MS/MS a simple 2 m/z window centered around the monoisotopic precursor would not do a great job of isolating the entire envelope for a charge 2 precursor. You would get M, M+1 and part of M+2. So, you would need wider than 2 m/z, and I am not quite sure how losing peaks beyond the first 3 would impact the fragment ion isotope distribution. Probably quite a lot for large precursors where you may not see the most intense isotope peak until M+1 or even M+2.

So, it is complicated, and I decided to leave it unsupported at least for a while, until we get more experience with Targeted MS/MS and DIA. To do this successfully for DIA, would require a larger unused overlap region than the 0.5 m/z region suggested in the SWATH paper, which only covers signal drop-off and not preservation of M+ peaks. The original SWATH technique only guarantees coverage for the monoisotopic precursor peak.

Thanks for posting your question. It is an interesting topic, for sure. Just not one that Skyline has good support for yet.

warham responded:  2024-05-01 15:43
So it's been a while, is this still the case? It seems like it is. This would be particularly useful evaluating small molecules or modified peptides, anything where fragmentation quantitation would be illuminating, especially where the fragment chemical formula is known.

I understand the issue with the isolation window skewing the envelope but in the alternative where we know the chemical formula for the fragment and if skyline were to calculate the theoretical isotopic envelope, as it does for precursors, this would actually help assess how much skew the quadrupole isolation window was creating.

Thanks as always for this wonderful wonderful tool
Nick Shulman responded:  2024-05-01 16:04
No, Skyline does not know how to monitor anything other than the monoisotopic m/z for fragment ions.
There is a discussion of the issues involved here:

I believe that we haven't actually found any datasets where this would improve the numbers.
-- Nick