Targeting M+2 for large peptides

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Targeting M+2 for large peptides paul grimsrud  2025-08-19 12:29
 

When working with very large peptides (~4 kDa), it seems that setting up a PRM method to isolate around the M+2 will help with signal intensity. In order to make Skyline import the chromatogram, I've added one 13C as an "Isotope Heavy" label at each termimus. In reality, however, the heavy isotopes are randomly distributed. What is the best way to handle this?

 
 
Nick Shulman responded:  2025-08-19 13:18
There was a bit of a discussion about targeting things other than the monoisotopic fragments here:
https://skyline.ms/announcements/home/support/thread.view?rowId=37846

I do not have any practical experience using a mass spectrometer, but it might be that you should widen the isolation windows in your acquisition method so that all of the ions from M+0 to M+2 are included.
If you do that, then the isolation window will probably be centered at M+1. If you do that, then you might have to change a couple of your settings in Skyline so that Skyline correctly associates the spectra targeting the M+1 m/z with the intended precursor in your Targets tree.

If your "Acquisition Method" on the Full Scan tab at "Settings > Transition Settings" is "PRM" then you should make sure that the "Method match tolerance m/z" on the "Instrument" tab is high enough so the center of your isolation windows still include the monoisotopic mass.
Alternatively, you could change the "Acquisition Method" to "DIA" and use the Isolation scheme "Results Only" and Skyline will realize that monoisotopic m/z is included in the isolation window.

It would probably be difficult to get the thing that you are proposing to work in Skyline where you have a separate Precursor for the M+2 m/z.
-- Nick
 
paul grimsrud responded:  2025-08-19 13:32
Thanks Nick! Changing the "Method match tolerance m/z" to the max of 0.6 (from 0.055) did the trick. Since my precursor is +4, the M+2 is less than 0.6 from the M+0. Got rid of the isotopic modifications, which sort of worked but was definitely wonky.