Can you send me the files "DKYF_SQ_20230803_KS480_Y_A549_400UG.raw" and "DKYF_SQ_20230803_KS480_Y_MKN45_400UG.raw"?

You can upload them here:
https://skyline.ms/files.url

I do not know why things look the way that they do in Skyline.
If you right-click on the chromatogram and choose "Transitions > Single" then you will be able to see a chromatogram when you select a transition in the Targets tree.
But, when Skyline is trying to show you the chromatogram for more than one transition, the graph ends up being blank, but I do not know why that is happening. I think it has something to do with there being very few points in the light chromatogram.

It will probably make more sense to me after I see your raw files.
-- Nick

Hi Nick，
The two data have been uploaded，the location is shown in the attachment.
And I try like this "If you right-click on the chromatogram and choose "Transitions > Single" then you will be able to see a chromatogram when you select a transition in the Targets tree." But I still cannot see the chromatogram of light peptide.
Could you take a picture？

thanks，

Kaylee

Thank you for sending those .raw files.
The reason that the chromatograms for the light peptide do not show up is that those chromatograms have only two points in them, and Skyline has trouble displaying a SureQuant chromatogram with only two points in it.

I think the underlying problem is that the heavy peptide was not really seen by the mass spectrometer. Your SureQuant method tells the mass spectrometer that when it sees the heavy peptide it should start collecting spectra for the light peptide. My understanding is that the heavy peptide is supposed to be spiked into your sample in a large enough quantity that it is very easy for the mass spectrometer to detect.

When Skyline extracts chromatograms for a SureQuant experiment, Skyline tries to figure out the time range over which MS2 spectra were collected for the light and heavy peptides. Skyline hides the parts of the chromatograms where the light and heavy peptides were not both being selected for fragmentation.

If you want to see what the chromatograms would look like if Skyline was not hiding the parts of the chromatogram where the mass spectrometer was not collecting spectra, you can go to "Settings > Transition Settings > Instrument" and uncheck the "Triggered chromatogram acquisition" checkbox. Then you can tell Skyline to extract the chromatograms again by pushing the Reimport button on "Edit > Manage Results".
When I did that with your data, it looked to me that your heavy peptide was not present in your sample.
-- Nick