Can you send us the files "04152024_HWAP8_LAMP1_NOTCH2_HHL_schedul_21min_R1_1.wiff" and "04152024_HWAP8_LAMP1_NOTCH2_HHL_schedul_21min_R1_1.wiff.scan"?
Files which are less than 50MB can be attached to these support request. You can upload larger files here:
https://skyline.ms/files.url
When you export a method for CE optimization, the method will contain lines for a different optimization steps according to the "Step size" and "Step count" settings that you see when you choose "Edit Current" in the "Collision Energy" dropdown on the "Prediction" tab at "Settings > Transition Settings".
In your document, "Step size" is set to "3" and "Step count" is set to "7".
If you choose "Edit current" in the "Optimization library" dropdown, you can see that many transitions have a collision energy value which is less than 21. For those transitions, you would not expect an exported method to contain the full complement of optimization steps because the collision energy value cannot be a negative number.
For the y5+ transition, the collision energy value is 19.84, so I would have expected that there would be 14 optimization steps ranging from step -6 with collision energy 1.84 to step +7 with collision energy 40.84.
The number of optimization step chromatograms that you are seeing for that transition is less than that, so I am not sure what is going on.
For Sciex instruments, CE optimization methods will have sets of chromatograms with identical Q1 values and with Q3 values that differ by 0.01. If there are fewer than the full complement of optimization steps (i.e. 15 chromatograms per transition) then Skyline will assume that the SRM chromatogram whose Q3 value most closely matches the product m/z of the transition in the Skyline document is the chromatogram for "Step 0" then assigns the other chromatograms step numbers based on their relation to that middle one.
After I see your .wiff and .wiff.scan files I might have a better idea of what went wrong.
-- Nick