Distinguishing between heavy carbon and heavy hydrogen

Distinguishing between heavy carbon and heavy hydrogen molly hopper  2024-02-22 11:24

I'm working on a small molecule tracing experiment where we've added a duterated molecutle (D5 Glutamate) as in internal standard in our study, and have a heavy labeled carbon tracer. We're trying to trace the heavy carbons, and in an unlabeled control, we've noticed a slight signal for [M5C13-H]. I am interpreting this as an issue with the masses being so close that skyline believes them to be the same peak. We have the resolution to see the difference between [M5H2-H] and [M5C13-H] on the instrument (Thermo Exploris 240). I'm hoping for some help with how we can resolve the peaks in skyline, as we're soon to start a study that uses C13N15 tracers.

Current Transition Settings:
MS1 Filtering, precursor mass analyzer: Orbitrap, resolving power: 240,000 at 200 m/z
Instrument: Method matcho tolerance 0.055 m/z.

I've tried changing both of these settings with no change to the integrations.

Windows 10, Skyline 64-bit


Nick Shulman responded:  2024-02-22 11:36
Can you send us your Skyline document and one or more of your .raw files?
If I could see exactly what your data looks like I might be able to give you a better answer.
Also, I am not sure I understand what your isotopic modifications but I expect that the Skyline document would make it clear.

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 gives you the option to include the .raw files or you can upload them to us separately.

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

If you are worried about signal spilling over between your isotopes I would recommend that you click on a point along the extracted chromatogram to bring up the spectrum viewer. The spectrum viewer will highlight the channels around the transition m/z values that Skyline summed across when extracting the chromatograms. The width of those channels is determined by the resolution or mass accuracy settings at "Settings > Transition Settings > Full Scan".
-- Nick
molly hopper responded:  2024-02-22 12:00
Thank you Nick,
I am working on geting a copy of the file to share that only has the relevant transitions/molecules.

Where in the Transition Settings > Full Scan can we define the mass accuracy? I only see option for resolution.
Nick Shulman responded:  2024-02-22 12:11
In the Transition Full Scan Settings, you will see "Mass Accuracy" if the selected mass analyzer is "Centroided". If your mass analyzer is something else such as "Orbitrap" then the setting will be called "Resolution".

It looks like you did not attach anything to this support request. That often happens if the file you were trying to attach is larger than 50MB.
If your attachment is too large you can upload it here:
-- Nick
molly hopper responded:  2024-02-22 12:16
I uploaded the document to the given URL. The file is named with Hopper Heavy Label
Nick Shulman responded:  2024-02-22 13:24
Thank you for sending your Skyline document.
I am not sure that I understand what you are doing with your heavy labels.
Your molecule of interest has the chemical formula "C5H9NO4" and you have six different Molecules in your targets tree, and each of those Molecules has a different number of heavy carbons from 0 to 5.

We do not normally set things up like that in a Skyline document because when they are separate Molecule's Skyline does not know that they are expected to have exactly the same retention time.

What you would normally do is have a one Molecule with the chemical formula C5H9NO4, and there would be two different Precursor's under that molecule, where one Precursor is light, and the other Precursor is heavy. The heavy Precursor would have 5 heavy carbons in it. The symbol that Skyline uses to represent a heavy carbon is:
The heavy modification that is applied to the light molecule to turn it into the heavy molecule is:
(that is, add five heavy carbons and subtract five regular carbons).

Skyline knows that a certain percentage of your light precursor are going to have one heavy carbon. These other isotopes are represented as Transition's under the Precursor. You can control how many of these isotope Transitions you get with the "Isotope peaks included" setting at "Settings > Transition Settings > Full Scan".
Skyline also knows that a certain percentage of your five heavy carbon precursor are going to only have four heavy carbons because it's difficult to manufacture isotopically pure reagents. You can control the expected isotopic purity with the "Isotope labeling enrichment" setting at "Settings > Transition Settings > Full Scan".

I would have used a transition list like the text file that I have attached here.

This would not really allow you to monitor all of the same m/z values. Skyline will not let you monitor an isotope m/z whose predicted abundance is less than 1% of the most abundant isotope. But, it would enable you to extract chromatograms for 146.0459, 147.0489, 150.0593 and 151.0627.

Does this help? I am not sure I understand how you have labeled your molecules and what information you are trying to get regarding partially labeled molecules.
-- Nick
molly hopper responded:  2024-02-22 14:36
Your explanation makes sense, but isn't going to give us the data that we need for our experiment.

This is a metabolomics tracing study. Our model organism is exposed to heavy glucose. We then monitor which metabolites the C13 carbons are incorporated into. In the case of glutamate, a different number of C13 labeled carbons may be present depending on the amount of time since labeled glucose exposure, so we need all of the isotopes.

Additionally, we add D5-glutamate as an internal standard to every sample to monitor instrument response in each injection.

D5-Glutamate and 5C13-Glutamate have very similar masses, and we seem to be having difficulty isolating each from the other in skyline, as evidenced by the presence of 5C13-glutamate in one of our samples that had no heavy glucose exposure (our process control). We are able to resolve them experimentally.

Perhaps we can set up a zoom call to discuss this further? Thanks for your help with this!
Nick Shulman responded:  2024-02-22 15:08
Have you tried clicking on a point along the chromatogram to bring up the "Full Scan" graph?

I am not sure if I am looking at the correct replicate, but what I see is that the peak for the D5-glutamate is ten thousand times larger than the peak for 13C5 glutamate.
It does not look like any of the signal from the 151.0773 (D5) mass is spilling over into the channel that Skyline is summing across for the 151.0627 (13C5) chromatogram.

I am not that familiar with the isotope distributions of all of these elements, but I wonder whether those tiny peaks to the left and right of the really big one might be caused by some other low abundance combination of 13C, 15N, 2H and even 18O.

I will send you an email.
-- Nick