|How to analyze and quantify multiplexed peptides (Q Exactive PRM) with Skyline?||gillespiekevinp||2020-04-09|
I'm having a lot of trouble with analyzing my RAW files generated by a Q Exactive HF.
I am only trying to analyze one protein. In my "Inclusion List" I constructed with the Thermo software, for each peptide, I have Multiplexed multiple precursor charges. But I have the heavy internal standard peptides and light peptides Multiplexed separately.
Using Xcalibur Qual Browser, I check for the precursor ions of these peptides (Full Scan) or the product y and b ions (DIA), and my peaks are present and reasonably intense.
However, I can't figure out the correct Transition Settings in Skyline to reflect any of this Multiplexed PRM data when I import it. And I can't figure out how I would generate and export the ratios of either Light/Heavy precursor ions or Light/Heavy MS2s from Skyline.
Any tips? I can attach files as needed to clarify. I've gone through tons of tutorials by this point, and any additional help would be appreciated.