Hi Folks!

I am utilizing Skyline Daily (23.1.1.268) to analyze my QQQ data, where I aim quantify relative abundance of different intracellular metabolites. During sample processing, I have included an internal, isotopic standards for relative & surrogate normalization purposes. With Skyline, I have successfully imported a transitions list with the appropriate heavy/light formulas, set up a normalization method, and specified which isotopic standard should serve as a surrogate. However, I'm curious how Skyline is actually quantifying the peak area ratio to the defined surrogate.

In my transition list, the majority of my molecules (including my isotopic standards) have multiple product ions and I have not specified as qualitative vs. quantitative. But, when I examine a Document Grid displaying the normalized areas, there is only a single value despite multiple transitions. Does Skyline have a default way of setting a quantitative ion that I'm overlooking? Or, without distinguishing qualitative vs. quantitative product ions, does skyline sum all available product ions and use this "total peak area" for the calculation?

Attached are screenshots to help clarify what I'm describing above. Let me know your thoughts & recommendations!

Thanks in advance,
Megan