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.

Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url
-- Nick

Hi Nick,

I have attached the skyline document below.

Thank you for your help

It sounds like you intended to attach a file but nothing actually got attached.
This often happens if the file you were trying to attach was larger than the 50MB limit for attachments.

You should try uploading your file here instead:
https://skyline.ms/files.url
-- Nick

It looks like the file you uploaded was zero bytes long.
I will send you an email directly in case it is easier for you to send files that way.
-- Nick

Thank you for sending that Skyline document.

I see that you have heavy and light things but those things are in separate molecules and they should separate precursors under the same molecule instead.
I was able to see the transition list that you used by going to "View > Other Grids > Audit Log".
In your transition list you had different values for the "Molecule Name" for your heavy and light molecules (e.g. "Acetyl-CoA(M+0)" and "Acetyl-CoA(M+2)").
What you should have done is have the same molecule name for your heavy and light ("Acetyl-CoA").

I have fixed this all in the attached "FixedTransitionList.txt". I have attached my Skyline document "HeavyAndLightPrecursors.sky.zip".

I was not what to do about the molecules like Malic acid where you have four heavy precursors with between one and four heavy carbon atoms. Those molecules are not included in my document. We might be able to help you with these if you could tell us a bit more about what's going on with them.
-- Nick

Hi Nick,

Thanks for your help. We treated cells with uniformly labelled glucose and now we're trying to track the incorporation of the 13-carbon. For the glycolytic intermediates we except them to either be completely labelled or unlabelled no in-betweens. For the TCA intermediates, it is a bit more complex and intermediates can have more possible isotopologues. This is why for molecules such as malic acid and citrate we had annotated all heavy precursors as "heavy". Do you think this is the best approach?

Also, would editing the transition list mean we would have to re-adjust the peak-integration? or would the current peak integration be maintained?

Best,

Mikky

For those molecules that have multiple "heavy" forms, the easiest thing might be to tell Skyline that they are all "light" precursors with different adducts.
That would mean that Skyline will not calculate the ratios for you-- you'd have to do that somewhere else such as in Microsoft Excel.
The only other thing I could think of to do would be to go to "Settings > Molecule Settings > Labels" and add a bunch of other label types such as "heavy1", "heavy2", etc. but I think that would be too complicated.

One thing that you should be sure to do is make sure that the right things are checked in the "Internal standard types" checkbox at "Settings > Molecule Settings > Labels".
Skyline assumes that the internal standard will be much easier to detect than the non-internal standard so that if a molecule has both a regular precursor and an internal standard precursor, the peak detection will only be based on the internal standard's chromatograms.
If your label types are equally easy to detect then I would recommend unchecking all of the boxes at "Settings > Molecule Settings > Labels".

The peak boundaries will all get lost if you change the Name of the molecules like I have done in the attached transition list. If you leave the name exactly the same for the light molecules and change the heavy molecule name to match the light molecule name then I think the peak boundaries will end up being preserved so long as you reimport by using the "Reimport" button on the "Edit > Manage Results" dialog.

If you are going to be renaming your molecules you could export the peak boundaries before you do that, and then import the peak boundaries later.
That is, you can do "File > Export > Report" and export the report called "Molecule Peak Boundaries".
Then, after you import your transition list into a new document and extract chromatograms you can edit the exported peak boundaries file and fix up the molecule names. You should also delete the column "Precursor Adduct" from the exported peak boundary file since you want the peak boundaries to be the same for all of the precursors under the same molecule.
Then, you can use the menu item "File > Import > Peak Boundaries" to read those peak boundaries into the new document.
-- Nick

Hi Nick.

Would it be appropriate to export reports with total MS1 area and the normalization divisor which I could divide the intensities by in R. I am trying to normalize the intensities by total ion current. Would that be an appropriate workaround?

Best,

Mikky

Yes, that sounds like a good idea.
Usually when people have both heavy and light things they are interested in the ratio between the light and heavy in which case normalizing by total ion current would not make a difference.
But, it sounds like you are hoping to measure something different.
-- Nick