Can you send us your Skyline document and your raw file?

In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and extracted chromatograms.

If that .zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:
https://skyline.ms/files.url

It would be helpful to see your Skyline document and your raw file (or, if it's a SCIEX instrument, then your .wiff file and the .wiff.scan)

There are a bunch of rules that Skyline uses to decide which SRM chromatograms in the raw file belong to which precursors and transitions in your Skyline document.
First, the SRM chromatograms are put into groups based on their Q1 value. Then, for each group whose Q1 value is within the mz match tolerance of a particular precursor in your Skyline document, Skyline looks at how many of the Q3 values match the m/z of one of the transitions under that precursor. Then, Skyline decides that the group with the most transition matches is the group that matches.

There are other things that get looked at too. If two SRM chromatogram groups have the same number of matches, then Skyline picks the group whose m/z's match closer. Also, if you specify the Explicit Retention Time on the precursor in your Skyline document, that number will sometimes be used to decide between groups of SRM chromatograms.

We will be able to tell you exactly what is going on when we see your data.
-- Nick

Thanks Nick for your swift reply.

Requested files uploaded:
200702_Lipid_sMRM_shared.sky.zip

-Thomas

Thank you for sending those files.

Your raw (.d) files each contain three chromatograms whose Q1 is 774.6.
They are:
SRM SIC Q1=774.6 Q3=184.096
SRM SIC Q1=774.6 Q3=184.097
SRM SIC Q1=774.6 Q3=184.0971

In your Skyline document, your transitions have product m/z's that are 184.1, 184.101, and 184.1011
So, for each of the transitions, the chromatogram that is closest to it is 184.0971, so that is the chromatogram that Skyline assigns to each of those transitions.

If you were to create add different transitions with m/z's that exactly match that, then Skyline would correctly give each transition a different chromatogram.

It sounds like the Q3 values that I am seeing in your Agilent .d file are different from what you expect to be in there.
I am using ProteoWizard SeeMS.exe to look at the chromatograms inside that file.

I am not sure why the Q3 values in your .d file are exactly .004 less than what you seem to be expecting. Let us know if you think there is something that Skyline is doing wrong.
-- Nick

Thanks Nick.

The Q3 value difference of .004 is really weird. It does not show up in the acquisition method nor when I open the data file in Agilent software.
I have created two transition lists, one for acquisition and one for data import into skyline. Import works now :)

I guess we are not getting away w/o product ion offsets for isomers in scheduled methods?

-Thomas