Skyline does not work for direct infusion data.

One reason that Skyline does not work is the way that Skyline calculates background by looking at the height of the peak at the beginning and end of the integration boundaries.
You can see how Skyline calculates background here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tip_peak_calc

In theory, you could tell Skyline not to do background subtraction by checking the "Triggered chromatogram extraction" checkbox on the "Instrument" tab at "Settings > Transition Settings".
However, Skyline would still end up doing a bad job choosing peak boundaries because Skyline really expects chromatographic peaks to have a decent peak shape.

That said, we would still expect Skyline to display a chromatogram for you.
If you send us your Skyline document and .raw file we could tell you why Skyline is doing what it is doing.
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
The Share Document dialog also gives you the option to include the raw files or you could send them to us separately.
Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url
-- Nick

Hi Nick,

Thank you for your help, please see attached the skyline file (QE_HF_PR1969_AMB_Peptidelist_10062025.sky) and the Raw files (PR1969_4EBP1_Rawfiles_10602025) to the file sharing support link.

As you can see I have uploaded the raw files into skyline. There are no matching spectra appearing on the intensity/RT plot yet I know there are matching spectra as when I have highlighted the RT 0-1 minute most of the precursor and fragments go green suggesting they are present. Perhaps there is a setting I am missing as to why I can't see the spectra.

Many thanks,

Sam

Thank you for uploading those files.

I see that "Triggered chromatogram extraction" on the "Instrument" tab at "Settings > Transition Settings" is checked.

"Triggered chromatogram extraction" does have the effect of telling Skyline not to do background subtraction but its primary purpose is to be used with acquisition methods such as SureQuant where the mass spectrometer has been told to only start collecting certain types of spectra when something else is seen in another spectrum.
When "Triggered chromatogram extraction" is checked, Skyline tries to detect the time range over which data was not being collected for particular precursors. In your .raw files, the time ranges over which the MS1 spectra were collected do not overlap with the MS2 spectra, so Skyline ends up thinking there was no valid time range over which the instrument was acquiring spectra that you would want to see.

One way to fix this problem would be to uncheck the "Triggered chromatogram extraction" checkbox.
A different way to fix this would be go to the "Full Scan" tab at "Settings > Transition Settings" and change "Isotope peaks included" to "None" which would tell Skyline to ignore MS1 spectra.

After you make either of these changes you would need to tell Skyline to extract chromatograms again such as by using the "Reimport" button on the "Edit > Manage Results" dialog.

Either of these changes would allow you to see chromatograms in Skyline. However, you will probably notice that Skyline does a poor job of peak picking with this direct infusion data, so you probably will want to use other software.
-- Nick

Hi Nick,

Thanks for the help, I can now see the chromatogram but given your advise I am also running a PRM method with LC-MS analysis to see if I can get better peaks to confirm the presence of my peptide.

many thanks,

Sam