Thomas,

Can you send us a .sky.zip file instead of the .sky file that you attached.

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 and spectral libraries.

That "File > Share" menu item is the best way to create a file that you can send to other people because Skyline makes sure that the .zip file contains all of the necessary files for the recipient to be able to open the document and browse the data.

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

Sure, sorry.

Thanks for attaching that Skyline document.

One thing I see is that you have chosen "DDA" as the MS/MS filtering Acquisition method on the "Full Scan" tab at "Settings > Transition Settings". When the MS2 acquisition method is "DDA", Skyline treats all MS2 chromatograms and non-quantitative which is why they are displayed as dotted lines. It sounds like that is what you intended.

I see that you have a data file called "ISTD only (Zero)_B-D3_2_4366.d" whose sample type has been set to "Standard" and whose Analyte Concentration is "1".
Why is the name of that file "ISTD only"? An external standard (something whose "Sample Type" is "Standard") should contain the internal standard as well as a known amount of the analyte so that unknown samples can be compared against it.
If the sample contained only the internal standard, its sample type would usually be set to "Blank".

I am not sure that I understand the rest of your question. Can you post maybe a screenshot?
-- Nick

Dear Nick,

I get that my explanation was a bit confusing, so now I will try to put it in an understandable way.

For now, I would like to quantify my undeuterated analytes with the surrogate standard approach.

The Skyline file I attached now is a different one, with (I think) the correct settings.
In this file, a dilution series 1:1 for 4 times of a deuterated Lipid mix is measured, named ISTD. The other measurement contains a system sytability test, which has the corresponding undeuterated analytes, samed SysSuitTest. I don't think it matters that later I would like to use the deuterated Lipids as internal standard. I added the concentration multiplier to account for the correct concentration in which the heavy standard was measured, like it was explained in the tutorial.
In the "Reports" tab of the dokument grid, I set the deuterated standard as "Standard", because there is no surrogate standard option. In the "Molecule Quantification 20250212 TP" tab i definded the concentration. Since I had problems, when the heavy analyte was in the same molecule as the light one, I split the heavy deuterated analyte from the light one and set it as surrogate standard. Maybe you can tell me, if that was necessary after we found out, what the problem was. As far as I understand it, I can't normalize to heavy if I follow this approach, which would be a drawback.

Now the concentration of the Standard is set, and I would like to produce a calibration curve. Since there are 4 replicates named as standard, I thought it might work. But it did not. What went wrong there? It says: "Error: All of the external standards are missing one or more peaks"

The second problem is even harder to explain for me. It's the same challenge that caused the splitting of the heavy from the light analyte. I will attach a screenshot to make it clearer. Since I have different replicates, I don't want the same analyte to be treated the same for every replicate. In this example here, the deuterated analyte should be used in the 4 "ISTD" replicates for my surrogate calibration curve, but should not be used as a standard in my SST measurement. But now I have the problem, that if I change the settings of an analyte for a single replicate (yellow highlight in screenshot), all other replicates will be changed as well. For example, if I change the standard type of SE27:1/16:1 heavy to surrogate standard, all 5 replicates will have that label and I can't remove it from my unknown (which in this case is the SST) without changing back the other replicates as well. If I want to calibrate now, with Regression fit "Linear", it uses the "standard" values for the light analyte, which is basically blank in my standard measurements, because they only contail the heavy analyte.

To sum it up in one sentence: I would like to treat Analytes diffently, if they are mesures in different replicates, is that possible?

I hope that my problems are clear and you have a solution for it.
All the best
Thomas

If you want to treat your replicates differently like that you would need to have two separate Skyline documents.
Different replicates within the same Skyline document cannot have different normalization settings.
-- Nick

Thanks for the fast reply!
Then I will refer to a different software for that part.

Have a nice day.

All the best
Thomas