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

Hi Nick,

thanks so much for your quick reply and the details. I'm attaching the .wiff files you requested. Hopefully they will help you figure out what's going on. :-)

~Regine

Thank you for sending those .wiff files.
I see that there is a chromatogram in there with Q1=445.722 Q3=732.367 and another with Q1=445.722 Q3=732.378
The difference between those two Q3 is 0.011.
That difference is allowed to be between 0.009 and 0.011 which means that it's right on the edge of what Skyline will accept. In this case, whether or not it's acceptable depends on the way that computers represent decimal numbers, and so it depends on exactly which numbers were subtracted from each other.

Do you have any idea why those numbers have been truncated to 3 decimal places? When I export a transition list those numbers go out to 5 decimal places?
If this is something the mass spectrometer did, we could certainly relax the restriction so that a difference of 0.011 is always acceptable to Skyline.
-- Nick