Hi, Thomas,

Can you send us your Skyline documents from your two computers?
In Skyline, youc an use the menu item:
File > Share > (complete)
to create a .zip file contaning your Skyline document and supporting files, including extracted chromatograms.

If those .zip files are less than 50MB, you can attach them to this support request.
Otherwise, you can upload them here:
https://skyline.ms/files.url

We might be able to figure out what is going on by looking at those .zip files, or you we might ask you to send us the raw files too.

Thanks,
-- Nick

Hi Nick,

thank you for your quick response. I attached the files. However, I set up the documents on both computers today from scratch again and now I got the same import results on both computers. So probably I somehow messed up the file generated on the windows 7 computer yesterday. However I still couldn't figure out the cause.

Best regards,
Thomas

Hi, Thomas,

It looks like with your Windows 7 file, you did not have "Centroid" selected under
Settings > Transition Settings > Full Scan > MS1 filtering > Precursor mass analyzer

The setting is currently set to "Centroid" in that file, but at the time that the chromatograms were extracted, I think it was something else.
(When you extract centroided chromatograms, Skyline add "?centroid_ms1=true" to the end of the file path in the document grid on the column "Replicates > Files").

So, your Windows 10 file used the Thermo centroiding algorithm before extracting the chromatograms, whereas on your Windows 7 copy, Skyline just summed the intensities in the profile data across the m/z channel.

I believe with centroiding turned on, we do get the same numbers as XCalibur does. Without centroiding turned on, we get numbers which are supposed to be proportional to what you get in other tools, but, effectively, the units are different.
-- Nick

Dear Nick,

thank you very much!

I tried to re-import (edit > manage results) the raw data with the centroid, 10ppm setting, but the peak areas did not change. Obviously the settings were changed and saved in Skyline, because it says "<transition_full_scan precursor_isotopes="Count" precursor_isotope_filter="3" precursor_mass_analyzer="centroided" precursor_res="10">" if I open the file in notepad++. If I remove the files in the manage results editor and re-import the raw-files from scratch I get the same intensities as in the windows 10 file, which should correspond to the "correct" centroided intensities.

I always thought it is okay to use the manage results editor to re-import the ion chromatograms after changing anything in the transition settings. Is this a bug or should I always import the raw-files from scratch if anything is changes in the transition settings?

I attached both files:
- re-imported in manage results editor
- deleted in manage results editor and re-imported from scratch

Best,
Thomas

That is a very good point.

Yes, it appears that we have a bug here. When you do a "Reimport", Skyline does not pick up the new "centroided" setting from the Full Scan Transition Settings. This is a consequence of the way that the centroiding gets encoded at the end of the raw file path.

The only setting which does not change when you do a reimport is whether or not the data gets centroided before Skyline looks at it.
So, for instance, if the setting when the chromatogram was first extracted was "Orbitrap" with a resolution of "60,000".
Then, if you change it to "Centroided" with a mass accuracy of 10ppm, Skyline will extract chromatograms from non-centroided (i.e. profile) data, but Skyline will sum intensities across a m/z channel whose width is determined by the current ppm setting, rather than the original resolution.

So, for now, if you change either MS1 or MS2 centroiding, then you should remove the replicate, and then import it again using "File > Import > Results".

Thanks for pointing this out! I will try to fix it in the next version of Skyline.

-- Nick

Dear Nick,

thank you very much for the explanation and for spending time on this and your plans to fix this.

Best,
Thomas