Metabolomic isotope data- can only view chromatogram for the nominal mass

Metabolomic isotope data- can only view chromatogram for the nominal mass sarahmacpherson676  2019-08-06

Hi, I am looking at metabolomics isotope data using GC-MS with a QIT mass analyzer. I was initially able to get the results for each isotope using Skyline Daily. However, when I opened a new document with the same data, I was only able to view the chromatograms for the nominal mass. Not sure if there is something wrong with the software, as I was able to analyze the data before with the same settings.
Attached is the .sky file that works and doesn't work.
Thanks for your help

Nick Shulman responded:  2019-08-06
Thank you for attaching those files (even though the attached files have the extension ".sky", they really are zip files).

I cannot get Skyline to open the thing that is in "". When I try, Skyline gives me an error about the mass of some of the product ions not being what they are calculated to be.

I see that there is an audit log in there. I can see that you inserted a transition list which looked like this:
Precursor Name    Precursor m/z    Precursor Charge    Label Type    Explicit Retention Time
Pyruvic    174    1    light    8.728
Pyruvic    175    1    heavy    8.748
Pyruvic    176    1    heavy    8.707
Pyruvic    177    1    heavy    8.769
(your transition list is much longer, but this list is sufficient to cause problems in Skyline).

With the current versions of Skyline or Skyline-Daily, what you end up with after inserting this looks like the attached image. That is, you get precursors with the m/z's 174, 175, 176, and 177, but the "transition mz" ends up being 174 for each of those things.

This is definitely a bug in Skyline. Our small molecule expert is on vacation right now. He will be able to give us a better explanation when he gets back next week.

Even though this is a bug in Skyline, I think you will be able to do what you need to do if you structure your document more the way that Skyline expects.
If you want to monitor the monoisotopic, M+1, M+2, etc ions, those should all be "transitions" under the same "precursor".

If you know the chemical formula for your molecule, you should use that instead of the number 174. When Skyline knows the chemical formula, Skyline can predict the mass of the ions in the isotope distribution.

I believe there is also some way to tell Skyline to extract chromatograms for the precursor ions in an isotope distribution even if all you have are the m/z values, but I do not know how to do that. I would have thought it would have been possible using the "Add Transition" menu item on the right-click menu for the precursors in the Targets tree. However, it looks like anything you add like that will be a custom product ion which Skyline wants to extract from MS2 scans, not MS1.

Maybe someone else on this support board will have better answers.
-- Nick
sarahmacpherson676 responded:  2019-08-12
Thanks for your response Nick,
I have been trying those work arounds, but it would be great if you could fix that bug when your small molecule guy gets back.
Thanks so much!
Brian Pratt responded:  2019-09-03
Hi Sarah,

First, some background: when you give Skyline a transition list with molecules by the same name but different masses it assumes you're describing some kind of labeling scheme. As Nick says, if you want to see the isotopic distribution you just need to provide the chemical formula, with just one line per molecule in the transition list.

There is still a bug here, though: the document is internally inconistent as the error message indicates. The problem is that as written it's trying to declare different mz values for the same molecule with identical (non-labeled) adduct descriptions (all "[M+]" instead of "[M+]", "[M+1.0]", "[M+2.0]" etc). I can't imagine how it got into that state though.

But really, this is beside the point - you're not interested in labeling, you're interested in isotope distributions. Is it a problem to add a chemical formula column to your transition list?

- Brian
sarahmacpherson676 responded:  2019-09-19
Thanks for your help Brian.
My samples are TBDMS derivatized, so I am unsure of the molecular formulas for some of my metabolites.
Adding the molecular formula does fix my problem though.