I can't choose my quantification ion

I can't choose my quantification ion antonin padioleau  2022-10-11

Hi all,

I have a problem with my transitions. I work with GC-(EI+)-HRMS and LC-(ESI+/-)-HRMS data acquired in full-scan mode and I use the small molecule part of Skyline.

I created my excel sheat with 1 quantifier ion and 1 or 2 confirmation ions, the quantifier in the upper line of my sheat. When I imported this sheat with copy/paste, I selected all ions as precursor (it didn't work with precursor and product ions, no data in the chromatograms). Next, in the left side, for each molecule, my precursors were rearranged from the smaller mass to the highter.
My problem is that for all the molecules, the quantifier ion is automatically the first precursor ion, the smaller one, even if it's not the best choice. I tried to select the "Quantitative" choice only for my quantifier ion but, when it's not the smaller ion, the message "Error: All of the external standards are missing one or more peaks" appears in the calibration curve window. I can only quantify with the smaller ion...

I tried to change the monoisotopic and average masses of my molecules, which are always the mass of the smallest ion, to my quantifier ion mass but Skyline automatically adds the difference between the former masses...

How can I choose my quantification ion and confirmation ion in another way?


Nick Shulman responded:  2022-10-11
Can you send us 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:

By default, all transitions are used for quantification. Normally, if you wanted some transitions to not be used for quantification, you would right-click on particular Transitions in the Targets tree and use the "Quantitative" menu item to mark them as quantitative or non-quantitative. Alternatively, you could use the Document Grid to display the "Quantitative" column and set it that way.

If you are using "ratio to heavy" as the normalization method, then Skyline tries to match up transitions from the heavy and light precursors. If you have not provided names for the transitions, then Skyline does this matching up positionally. If you do not like the way that Skyline is doing this matching up you can go to:
Settings > Molecule Settings > Quantification
and check the checkbox "Simple precursor ratios".

I am not sure that I understand your question but after I see your Skyline document it will probably make sense to me.
-- Nick
antonin padioleau responded:  2022-10-12
Thank you Nick for your answer.

With the Skyline document it will be easier to see the problem for you. I uploaded my GC-HRMS file following the link.

For example, the DEET in the molecule list "ACN GC". Its quantifier ion is 190.1230 and its confirmation ions are 162.0910 and 191.1300. By default all the transitions are used as "Quantitative" with the menu item. But when I tried to mark the confirmation ions as non-quantitative the error message appear in the calibration curve window and in the report no concentration are calculated. The only way to have concentration result is to mark the lighter ion as quantitative.
This was confirmed when I compared if the peak areas in the "Calibration curve" and in the "Peak areas - Replicate comparison" windows matched. It's clear that the value in the calibration curve is for the area of the ion 162.0910.

I had the same problem with all molecule whose the quantifier ion is not the lighter one.

antonin padioleau responded:  2022-10-12
Dear Nick,

My colleague and I found what was wrong with my transition list. The Isotope label type was "light" for the lighter ion and "heavy" for the other. By marking all with "light" label I can choose my quantifier ion.

Thanks for your help,
Nick Shulman responded:  2022-10-12
Thank you for sending your Skyline document.
I see that for most of your molecules you have three different precursors in the Targets tree (one light and two heavy).
I am not sure exactly what Skyline does in a case like this, but I think things will work better if you arrange things so that there are only two precursors under each molecule, with maybe multiple transitions under each precursor.

For DEET, what is the relationship between the 162.0910, 190.1230 and 191.1300 ions?

(I am not much of an expert on small molecules, but I am hoping someone else might be able to give advice about how to set up the document for what you are trying to analyze).
-- Nick
antonin padioleau responded:  2022-10-17
Dear Nick,

The problem was that Skyline marked automatically the three ions with different isotope label. For exemple the three ions for DEET were marked light for 162.0910 and heavy for 190.1230 and 191.1300. If I undestood well, "heavy" label suggest that the ion is labelled with 13C or D so these ions are not considered for the quantification.
I changed the label to light and now I can choose the ion for the quantification.

For DEET, 190.1230 is the monoisotopic mass, 191.1300 is the mass with the contribution of natural 13C and 162.0910 is the mass of a fragment/product.

I tried to import my transition list with one precursor (my quantification ion) and two product ions (my confirmation ion) but after deconvolution of the raw data there was a message "No chromatogram data" or something like this.

Brian Pratt responded:  2022-10-17
Hi Antonin,

I would be interested to see your excel file, to see if there is anything Skyline could have done better in interpreting it. Or, at least, I might be able to offer some advice in formatting it in a way that Skyline will understand.

Best regards,

Brian Pratt
antonin padioleau responded:  2022-10-18

Here is the precursor list used (sheet "precursor only") and a .raw data acquired in GC-(EI+)-HRMS with all the molecues listed. Retention time are not updated as I changed my column before this injection.
I add the transition list I tried to work with. I think this solution didn't work because i. I acquired in full-scan (but EI breaks molecules) ii. I didn't enter collision energy.

Brian Pratt responded:  2022-10-18
Looks like those attachments failed, perhaps you can upload to http://skyline.ms/files.url ?


Brian Pratt
antonin padioleau responded:  2022-10-18
Here is the excel file. I can't upload the .raw on the file sharing page...

Brian Pratt responded:  2022-10-19
Thanks for sending that.

Taking just the example of (Z)-Dimethomorph:

On the "Precursor Only" sheet, Skyline reads that as a molecule of mass 165.055, with precursor ions having adducts [M+], [M136.007+], [M222.068+] where 136.007 and 222.068 are the mass of whatever is making those ions so heavy.

On the "Transition" sheet Skyline interprets this as a molecule of mass 301.062 with fragments of mass 165.055 and 387.123. This seems odd: a fragment larger than the parent? And, of course, the [M+] ion having a completely different m/z than on the other sheet seems suspicious.

If this is the molecule described at https://pubchem.ncbi.nlm.nih.gov/compound/Z-Dimethomorph then I'd think that 387.123 is the actual m/z for the [M+] ion, being as that's the monoisotopic mass of C21H22ClNO4.

How were these values produced? They seem a bit scrambled. I think that you're probably trying to describe fragments of m/z 301.062 and 165.055 from something with precursor m/z 387.123. That would be formatted like this:

Molecule Name,    Molecule List Name,    Retention Time,    Precursor m/z,    Precursor Charge,    Product m/z,    Product Charge
(Z)-Dimethomorph,    ACN GC,    23.86,    387.123,    1,    165.055,    1
(Z)-Dimethomorph,    ACN GC,    23.86,    387.123,    1,    301.062,    1

I hope this helps!
Best regards,

Brian Pratt