MRMHR on ZenoTOF for small molecules

MRMHR on ZenoTOF for small molecules viragsagikiss  2024-01-10 15:45

Hi all,

Apologies if this has been discussed before I normally work on low resolution targeted small molecule workflows.
I ran the MRMHR mode for small molecules a SCIEX ZenoTOF 7600 system, could you help me if there is a tutorial for that?
I used the small molecule tutorial combined with the PRM tutorial for high res instrument. I could create the transition list and read in the data but only MS1(Precursor ) data shows up, no fragments. I added 'f' in the transition list settings.

Thanks you for pointing me in the right direction,

Nick Shulman responded:  2024-01-10 15:53
Adding "f" in the Ion Types at "Settings > Transition Settings > Filter" is not going to help very much because, for small molecules, Skyline never knows how to predict what the fragments might be.
The usual way to tell Skyline what transitions you are interested in is with either the "Edit > Insert > Transition List" menu item or "File > Import > Transition List".
The Small Molecule tutorial (page 4) will show you how to create a transition list and insert it into Skyline:

Let us know if you get stuck anywhere.
-- Nick
viragsagikiss responded:  2024-01-11 03:06
Hi Nick,

Thank you for the answer. I have attempted to add a transition list just like you described above. (I know the expected fragments from the QQQ experiments and also i can look at the Full Scan data in Skyline.)
But there are no peaks at the transitionsonly at the precursor. - I have reimported the data files.

I attached a screenhot, tested a few things. I am looking at a mixture of 3 compounds here, hexosephosphates, i can see the peaks at the right retention time as MS1/precursor.

viragsagikiss responded:  2024-01-11 04:30
Hi Nick,

Just an update that now i can see now the fragments and the precursors together! (attached for malate)
It works for most of my compounds, but not the hexosephosphates.
I tried a few things in the Transition Setting Tab:
- anything i change in the Filter tab does not affect the result, not even if I use the wrong M+H/M-H, so i assume those parameters are not used.
- Library: i assume nothing i change here has an effect as i dont use Libraries?
- so the the only parameter i can imagine that affects the fragment ion extraction window is the Method Match Tolerance under Instrument Tab. I was hoping to increasing that helps but no luck.

Also, i dont seem to be able to get a list of MS peaks from the full scan results.

I keep you updated.
Virag and Yufeng
Nick Shulman responded:  2024-01-11 06:45
I cannot tell from your screenshots whether anything is going wrong, but if you send us your Skyline document and raw files we can always take a look.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms. The "Share Skyline Document" dialog also has the option to include the results files, which you can use, or you could separately package up the .wiff (or .wiff2) and .wiff.scan files.

Files which are less than 50MB can be attached to this support request. You can upload larger files here:

The "Full Scan" graph in Skyline was only really intended for diagnosing problems with the chromatogram extraction. You cannot get much data out of that full scan graph. If you are interested in looking at spectra, there are probably other tools that are better for that than Skyline, including perhaps some tools that came with your Sciex instrument.

By the way, if you are interested in exporting lists of numbers from Skyline such as chromatogram peak areas, you should take a look at the Custom Reports tutorial:

If you are interested in working with calibration curves in Skyline, I would recommend the Absolute Quantification tutorial:

If you are interested in comparing peak areas between groups of replicates (e.g. Healthy & Diseased), you should look at the Group Comparison tutorial:
-- Nick
-- Nick