Peak quantitation/ integration issue for high resolution small molecule data

Peak quantitation/ integration issue for high resolution small molecule data chevans  2021-10-26

Apologies in advance for what may be an elementary question. I’m trying to use Skyline for analysis of small molecule data collected on an Agilent TOF. I have a data set in which, for one or a few data files, the mass accuracy wasn’t as good as it should have been for a few scans (probable intermittent loss of reference ion due to co-elution with salts or other interfering species). Those scans fall in the middle of a few peaks I’m trying to quantitate; for example, acetylcarnitine. This particular peak has an ugly "split" appearance due to poor retention on the column, but Skyline handles it fine for most of the data, the issue only crops up when the mass accuracy for a few scans is poor. In those cases, skyline is integrating only one part of the peak. I’d like to widen the “mass extraction window” (not the "skyline" for it, I realize, but concept should be the same) so I can get integrate the whole peak. This works fine in Agilent Quant software. But no matter what parameter I try changing in the “transitions settings” menu, the split peak doesn’t change and the integration is wrong. The attached image illustrates the problem.

I could just use Agilent Quant for this project but I’m hoping to use it to analyze data on multiple different instruments and I like the “unifying” nature of Skyline software. Obviously an even better fix would be to solve the mass accuracy issue, but this crops up often enough that I'd like to be aware of a workaround, if one exists.

Thanks very much for your help!


Nick Shulman responded:  2021-10-26
I am not sure that I understand what kind of data you have.

Your question makes it sound like you are asking Skyline to extract chromatograms from full scan data, and you would like to tell Skyline to sum the intensities across a wider m/z channel in each spectrum. The setting to change that is one of the "Resolution" values on the "Full Scan" tab of the Transition Settings dialog.
The confusing part is that in your screenshot your Transition Scan Full Scan Settings are not telling Skyline to extract chromatograms from either MS1 or MS2 full scan data, so you must be talking about SRM data.

With SRM data, the setting which controls how closely the Q1 and Q3 values need to match the calculated precursor and product m/z's is the "Method match tolerance m/z" on the Instrument tab of Transition Settings. However, that setting controls whether a particular chromatogram in the raw file will be used by a particular transition in the Skyline document. That setting would never cause part of the chromatogram to drop to zero like that.

If you send us your Skyline document and your raw file we can probably figure this out.
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. Otherwise, you can upload it here:

You should also send us your raw file. It sounds like you have an Agilent ".d" folder. You should zip that up and send it to us.
-- Nick