Missing Transitions

support
Missing Transitions chrysopoulou  2023-10-26 03:05
 

Hi everyone,

We are running targeted methods with isotopes in QQQ-LC/MS (Agilent) and we experience missing transitions when importing the results in Skyline.

I am attaching the transition list that we inserted, some raw data, and a screenshot of the Skyline interface.

As it is the first time I am trying to troubleshoot in Skyline, please let me know if you need more details.

Best,
Maria

 
 
Nick Shulman responded:  2023-10-26 04:36
I used ProteoWizard SeeMS.exe to look at the chromatograms in your file "Pig1_KidneyMedulla_left_positiveControl.d".
I see that there are groups of chromatograms which are clearly supposed to be matched to multiple transitions under the same precursor in your Skyline document, but the Q1 values of those chromatograms are slightly different.

For instance, one of the precursors in your document has the precursor m/z 338.9900, and has two fragment ion transitions 241.0000 and 79.0000.
In the .d file, there is a chromatogram with Q1=338.99, Q3=78.996 and Q1=339.00 and Q3=240.996.

When Skyline is trying to decide which chromatograms in the raw file correspond to which transitions in the Skyline document, Skyline allows the Q1 and/or Q3 values to be slightly different than the precursor and or product ion m/z values. The amount that these numbers are allowed to be different is controlled by the "Method match tolerance m/z" setting at "Settings > Transition Settings > Instrument".

The "Method match tolerance m/z" value does control how much the Q1 value is allowed to be different from the precursor m/z in your Skyline document.
However, when Skyline is matching chromatograms in the raw file to transitions in the document, within any group of chromatograms which are to be matched to a group of transitions in the Skyline document, the Q1 values must be identical.

That is, when Skyline is trying to figure out which group of chromatograms should match to 338.99->241 and 338.99->79, Skyline is only able to consider the single chromatogram Q1=338.99 Q3=78.996 or Q1=339 Q3=240.996 because for these two chromatograms the Q1 values are slightly different.

I hope this makes sense.

I see that in TransitionList_TCA_all isotopes.xlsx, the precursor m/z values of the different transitions were also different (339 and 338.99), but it seems that Skyline has put both of those into the same Precursor in the Skyline document in a way that causes this problem for you.

I also see that in your transition list, for each molecule there are multiple precursors with masses +0, +1, +2, +3, and +6.
I assume the +0, +1, +2 and +3 precursors correspond to the isotope envelope of the unlabeled molecule, and the +6 precursor is a heavy labeled precursor.

In SRM experiments, for the unlabeled molecule, people usually only collect data for the monoisotopic Q1 value producing the monoisotopic Q3 value.
The reason for this is that all fragment ions from the monoisotopic precursor will have zero heavy atoms in them (because there were zero heavy atoms in the precursor).
For the M+1 precursor, some of the fragment ions will happen to have zero heavy atoms in them, and some fragment ions will have one heavy atom.
The mathematics of getting useful numbers out of the M+1 chromatogram ends up being really complicated so people usually don't acquire the data.

I hope this helps.

By the way, if you have more questions, it would probably be helpful if you could include your Skyline document.
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.

If that .zip file is less than 50MB you can attach it to this support request. You can upload larger files here:
https://skyline.ms/files.url
-- Nick