What have you tried so far? Have you had a look at any of the tutorials at https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials ? In particular the ones in the Small Molecules section?

Best regards,

Brian Pratt

I did read those, thanks! I'm still not finding how to set the parameters for the Transition Settings for my type of data. I will upload a couple screenshots of what I currently am trying which I have no idea if is correct...

My machine settings were:

Single-quad, Agilent 8890 GC, 5977B GC/MSD
Positive ion mode
Solvent delay: 5 min
m/z range: 45-650
Threshold: 150
Energy: 70

Thank you for your patience and help with a novice!

A large missing piece of this puzzle is the transition list - Skyline is for targeted mass spec, so knowing what you're looking for in the data helps make sense of what you've put in the settings.

Perhaps you can provide your Skyline document, and one of your Agilent files?

Use Skyline's File > Share menu item to create a .sky,zip file. You can upload everything to http://skyline.ms/files.url or by any other means if you need something more secure.

Best regards,
Brian Pratt

Thanks so much. I just uploaded a file named skyline_062525_share

OK, a couple of suggestions:

As your input transition list apparently provided no chemical formula information, Skyline can't generate isotope envelopes, but still I would set Transition Settings > Full Scan > MS1 Filtering as:
Isotope Peaks Included = 3, Precursor Mass Analyzer = Centroided, Count = 3.

Set MS/MS Filtering Acquisition Method as None - - this is MS1-only data, so the MS2 "Full Scan" settings have no effect.

Make those changes then use Edit > Manage Results to reimport the ,D files with the revised settings.

I tried that and now the data looks really weird. I will upload the new file. Here's a screenshot

I see it too - "QIT" was the proper setting, apparently, rather than Centroided.

BTW the .sky.view file is for saving Skyline window layouts, it's not a screenshot.

Thanks! Should it be set to "None" or "Count"? And I thought I had uploaded both a screenshot and the .view file sorry!

“Centroided” was the only error in my revised settings.

Thank you so much for your assistance! You're amazing and appreciated