Skyline for small molecules, full scan-MS1, DDA-MS2

support
Skyline for small molecules, full scan-MS1, DDA-MS2 stepan koudelka  2023-08-11 01:05
 

Dear Skyline support team.
I am using high-resolution Orbitrap Tribrid to analyse small molecules. For an individual chemical standard, a precursor is detected by full scan MS1 as well as fragmented via DDA-MS2 using isotope label free acquisitions.
Precursors and the five most intense fragments of a corresponding precursor are summarized to create a Transition List feeding Skyline (22.2.0.351) via Molecule Interface. For the graphs, I can monitor only precursor(s) Retention times, Peak Area, and Mass Errors after setting Full-Scan bookmark according to the used MS1/MS2 acquisition parameters. Are there any visualization settings and/or proper input format for the Transition List and the Transition Settings… to monitor these up to 5 fragments together with the corresponding precursor in a particular graph simultaneously (for the loaded results)?
Thank you.

 
 
Nick Shulman responded:  2023-08-11 07:48
I am not sure that I understand your question so if you send me your Skyline document I might be able to give you a better answer.
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.
If that .zip file is less than 50MB you can attach it to this support request. You can upload larger files here:
https://skyline.ms/files.url

Skyline is mainly a tool for extracting chromatograms.
Chromatograms which have been extracted from DDA data usually do not look good because they have long straight lines connecting the places where a matching precursor happens to have been selected by the mass spectrometer for fragmentation.
You can tell Skyline to extract chromatograms from DDA data by going to "Settings > Transition Settings > Full Scan" and choosing "DDA" in the "Acquisition Method" dropdown.
-- Nick
 
stepan koudelka responded:  2023-08-15 04:47
I´ve been loaded a zip. file related to the topic. Thank you.
 
Nick Shulman responded:  2023-08-15 05:37
Thank you for uploading that .zip file.

One thing that I should have mentioned is that after you change the "Acquisition Method" at "Settings > Transition Settings > Full Scan", you will need to tell Skyline to extract chromatograms again in order to see whether that change has an effect.
That is, if you already have results in your document when you change the transition settings, you will need to go to:
Edit > Manage Results
and then select all of your replicates and push the "Reimport" button.

It might be that if you did that you would be able to see some MS2 chromatograms in your document.

If pushes the Reimport button does not work for you, you should send me some of your .raw files and I will be able to give you a better answer about what is going on.
-- Nick
 
stepan koudelka responded:  2023-08-21 04:38
Examples of raw data were loaded. Thank you.
 
Nick Shulman responded:  2023-08-21 16:12
Thank you for sending those .raw files.

It turns out that if you have choose "None" for "Isotope Peaks Included" at "Settings > Transition Settings > Full Scan" and you choose "DDA" for the MS/MS Filtering Acquisition Method, then you will not get any chromatograms.

That is, if the Acquisition Method is "DDA" you have to tell Skyline to extract some MS1 chromatograms; otherwise you won't get any chromatograms at all.

I don't think we actually intended for this to be the behavior, but it happens to have worked out this way.
DDA chromatograms in Skyline are not very useful, because they typically have long straight lines connecting the places where the mass spectrometer happens to have selected a matching precursor for fragmentation.
We always imagined that DDA chromatograms in Skyline would only serve to give a human a little more understanding about what the mass spectrometer was doing in between collecting MS1 spectra.

If you change the "MS1 filtering Isotope peaks included" setting at "Settings > Transition Settings > Full Scan" to something other than "None", and then reimport your results you will see some chromatograms for your MS2 transitions.

Some of your transitions will still not end up with any MS2 chromatograms because there were no MS2 spectra matched the precursor. If you change the "MS1 filtering Resolving power" to a lower number, then there will be more MS2 spectra which match the precursors in your document.
-- Nick