• Sign In
  1. Requests

Question about how Skyline is handling MS2 data

support

View Request

view list print
Question about how Skyline is handling MS2 data zrhopkins  2024-07-03 05:19
 

Hi,

I have a question about how Skyline is pulling transition data from Thermo .raw files and I'm going to try and attempt what I mean but I can provide a skyline file to show what I mean. Some background I'm putting in Full scan ddMS2 data from an Exploris 120.

The issue we are seeing is for compounds that we have only a 2 m/z difference between the native and IS. For example, Native mass is 712.9437 and the IS is 714.9540. I've attached a picture for now to show what I mean. We have an injection for a blank and we see now peak for the native mass, but we see fragments appearing. We know the native and IS can produce similar fragments because of the low mass labeling.

So the question I have is Skyline just pulling an ion channel for transition and that means if anything produces that fragment mass at that time then it would be captured and shown? This would explain why we see fragments from the IS when there isn't any of the native present in the blank to produce those ions.

I've checked the same data in Thermo TraceFinder. When we look at the blanks there is no peak for the native and there is not fragments showing up.
 
 
zrhopkins responded:  2024-07-03 06:21

Maybe some of this issue is based on whether I'm using DDA or PRM in the software.

So we are producing Full scan ddMS data on a Thermo Exploris 120. We are looking to quantify on the MS1 peak and just use MS2 peaks for qualification. So what would you do when setting up your Skyline file based on that. Should one setup the MS/MS filtering as 1) DDA or 2) PRM?

I've noticed for this 712.9473 compound that fragments I was noticing in the blanks when there isn't a peak for the native compound disappear when I switch from DDA to PRM.
 
Nick Shulman responded:  2024-07-03 22:37
When the acquisition method is "DDA", a MS2 spectrum will contribute to the MS2 extracted chromatogram if the isolation target m/z falls within the MS1 m/z extraction channel.
The width of the m/z channel that Skyline sums across when extracting MS1 chromatograms is controlled by the "Precursor mass analyzer" and "Resolving power" (or "Mass accuracy") settings in the "MS1 filtering" section at "Settings > Transition Settings > Full Scan".
Note that the "Isotope peaks included" setting at "Settings > Transition Settings > Full Scan" does not affect the matching of MS2 spectra to precursors-- Skyline considers all of the precursor m/z's that would show up when you set the isotope peaks included to the highest possible number.

If you click on a point along an MS1 chromatogram, Skyline will show you an MS1 spectrum, and Skyline will highlight regions around the precursor isotope m/z values. If the isolation target m/z falls in one of those highlighted windows for the light precursor, then that spectrum will contribute to that light precursor's MS2 extracted ion chromatograms.

When the acquisition method is "PRM", Skyline compares the isolation target m/z to the monoisotopic m/z of the precursor. The "Method match tolerance m/z" setting at "Settings > Transition Settings > Instrument" controls how close those two numbers need to be.
-- Nick
 
zrhopkins responded:  2024-07-08 08:20
I guess I'm still a bit confused because there is no peak for the MS1 precursor mass but I'm getting peaks for the MS2 (I'm pretty sure these are coming from the matched mass labeled compound). If there is no peak for the precursor mass (712.9437) then I shouldn't be seeing fragments for the MS2?

If I click on the MS1 chromatogram where there is no peak it opens the MS1 spectrum and there is no peak at that ion, but there is a peak at the [M+2] ion (714.9540). (see attached).

Are you saying that because there is a peak in the MS1 spectrum for the [M+2] mass that the software will pull MS2 data for that [M+2] mass also? So if you have mass labeled compound that is only M+2 different than the light precursor compound and produces similar fragments as the light precursor then you would get chromatographic peaks in the MS2 for the M+2 fragments when there is no light precursor present?

If so is there a way to get the software to not do that?