Precursor Intensity from MS/MS spectrum

support
Precursor Intensity from MS/MS spectrum bseale  2021-04-09
 

Hello,

I have a PRM skyline file for 100 proteins and 300 peptides acquired on a Bruker timsTOF. I have particular selections for all the various transitions and when I add my results in everything appears to be detected etc. correctly. However, I wish to compare the intensity of unfragmented precursor transitions to the other selected transitions (b, y ions). When I add the various areas or intensities to reports the precursor transitions always report the total area of the chromatogram while the fragment transitions appear to report the expected (lower) value.

My question is simple. From the screen shot, how to do I get a report that will tell me the intensity or area (either is fine) of the MS/MS precursor (red) so I can compare it to those of the other fragment transitions (blue)?

 
 
Nick Shulman responded:  2021-04-09
If Skyline is extracting chromatograms from both MS1 and MS2 spectra, then the chromatograms for precursor transitions will be extracted from the MS1 scans.

One way to tell Skyline to extract precursor chromatograms from the MS2 scans instead would be to go to:
Settings > Transition Settings > Full Scan
and change "MS1 filtering isotope peaks included" to "None".

If instead you want Skyline to extract precursor chromatograms from both MS1 and MS2, then you have to trick Skyline into thinking that you have another transition in your document whose mass is the same as the precursor, but which needs to be extracted from MS2 spectra. The way to do that is to tell Skyline that you have a neutral loss whose monoisotopic and average mass is .0001. (Neutral losses are not allowed to have a mass of zero, so you have to make it .0001).

I can't find good web page with information about how to define neutral losses in Skyline, particularly loss-only modifications. You should go to:
Settings > Peptide Settings > Modifications
and define a modification which is going to apply to every peptide in your document (probably by saying that it applies to any C-terminal amino acid) and then use the "Loss >>" button to add a neutral loss with a mass of .0001.

I would recommend using Skyline-Daily instead of Skyline 20.2 if you want to use loss-only modifications. In Skyline 20.2, it was not possible to have a loss-only modification on a peptide that had any explicit modifications, but we have fixed that in the most recent Skyline-Daily.
-- Nick
 
bseale responded:  2021-04-09
Interesting! I see how it's all laid out now. Thank you for the help, I believe I've got what I need.