Basic normalization

Basic normalization jm007  2022-02-24

I am returning to Skyline after a few years, so I am a bit rusty on the basics, so please forgive my lack of current knowledge.
I am performing a small molecule analysis with standard curve and heavy compound spiked into standard curve, QC, and unknowns.
Current issue is I can generate the cal curve only when I have Normalization set to None. When I switch to Normalize for heavy, standard curve goes away. I am also a bit confused by the fact that each transition, be it heavy or light, gives me a ratio with a very large value- millions. What ratio could that be? I see I can right click on precursor or transition in the targets list, and see Quantitative- what is this and should it be on all transitions? For this analysis, I have three transitions for the unlabeled, but could only get one 'heavy' transition. Do I need matched sets of transitions for normalization to work?
I am obviously lost! Thank you so much and let me know if I can provide additional info!

Kind regards,

James Movius

Nick Shulman responded:  2022-02-24
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:

-- Nick
jm007 responded:  2022-02-24
Hi Nick!

Many thanks- here is this file. Thank you for letting me know where I went wrong so I don't do this again. I am going to be using SL for many of the projects here at the MedChem MS facility- Dale is a believer now!

Kind regards,

Nick Shulman responded:  2022-02-24

Thanks for sending that Skyline document.
The problem is that you have two separate molecules ("Melatonin" and "Melatonin d4"), instead of what you are supposed to have is one molecule with both a heavy and light precursor under it.

Here is a transition list which you can copy and paste into Skyline which will give you a single molecule (named "NewMelatonin") which will have a heavy and light precursor under it:

Molecule List NameMolecule NameMolecular FormulaPrecursor AdductPrecursor MzPrecursor ChargeExplicit Collision EnergyProduct MzProduct ChargeProduct Adduct

After I do that, I do end up with a calibration curve, but the slope of the calibration curve is negative, which either means that the Analyte Concentrations that you have set on your Replicates are wrong, or maybe the real normalization method was supposed to be "Ratio to Light". I could not figure out how to make your calibration curve look normal, so you might have to ask us some more questions.

By the way, if you wanted to set the normalization method to "Ratio to Light", you would have to first go to "Settings > Molecule Settings > Labels" and check the checkbox next to "Light" and uncheck the box next to "Heavy" so that Light becomes the internal standard. After that, you will be able to choose "Ratio to Heavy" as the normalization method at "Settings > Molecule Settings > Quantification".

-- Nick