Chromatogram information unavailable

Chromatogram information unavailable pedrofrancobh  2023-06-28 00:54

Dear Skyline team,

I am trying to build a PRM method for some target proteins based on synthetic peptides I ordered. I injected a mixture of these peptides in a matrix from a different organism and could get them detected using a DDA method. I uploaded the MaxQuant files as a library and the fasta file as a background proteome, so far it looked good, but when I try to add the DDA results I get an error that "chromatogram information unavailable", and can't do any extra work.

Attached is the Skyline session and I uploaded a raw file to your sharing server.

Thank you for the support!

Nick Shulman responded:  2023-06-28 08:19
When I look at your Transition Full Scan settings at "Settings > Transition Settings > Full Scan", I see that the "MS1 filtering Isotope peaks included" is set to "None".
With DDA data, the only chromatograms which are meaningful are the MS1 chromatograms, so you should change that setting to something else (either "Count" or "Percent").
After you have changed those settings you can tell Skyline to extract chromatograms by going to "Edit > Manage Results", selecting everything and pushing the "Reimport" button.

If you do that, you will get MS1 and MS2 chromatograms. The MS2 chromatograms will have long straight lines which is what MS2 chromatograms extracted from DDA data always look like because with DDA there are long intervals where a matching precursor has not been isolated by the mass spectrometer.

It seems like a bug that you did not get any MS2 chromatograms when you were not also asking for MS1 chromatograms. We probably never tested the scenario of not extracting MS1 chromatograms when the acquisition method was "DDA". I will try to figure out why Skyline is not behaving as expected.
-- Nick