MRM QQQ transition peaks not detected in Skyline

MRM QQQ transition peaks not detected in Skyline ylee  2024-03-26 14:34


I ran three different m/z from a compound as shown in the transition list attached on Agilent QQQ. I can see the peaks fine at retention time around 4min for two transitions and less than 1min for one transition from Agilent Quantitative Analysis software. However, when I load the same raw files onto Skyline, I do not see any peaks and the retention time window is shown only up to 1.2min for the chromatograms which does not cover where the peaks are supposed to be in. Even after changing the explicit retention time and window, the chromatograms stay the same and I do not see anything.
I have attached the transition list, raw files, and Skyline template.

Please let me know how I can go about resolving this problem.

Thank you.

Nick Shulman responded:  2024-03-26 14:45
You should go to:
Settings > Transition Settings > Instrument
and change "Method match tolerance m/z" to a higher number such as "0.2".

The problem is that your current value of that setting is 0.055.
The precursor m/z in your Skyline document is 304.3148 but the Q1 value in your .d raw file folder is 304.2.

I used SeeMS.exe from ProteoWizard to see the list of chromatograms that were present in your .d folder.
-- Nick
ylee responded:  2024-03-27 13:17
Thank you! I see the peaks now.