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Incorrect quantification of low-intensity MRM peaks close to background noise

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Incorrect quantification of low-intensity MRM peaks close to background noise ioanna kyritsi  2026-01-20 01:00
 

When a peak has very low intensity (close to background noise), I observe incorrect quantification in Skyline, but only for compounds present at very low concentrations. In these cases, the QC samples fall outside the acceptable range. However, when I process the same MRM dataset using another LC-MS software, the QC values are within the expected range.

What could be causing this discrepancy in Skyline (background subtraction, noise threshold?), and which settings or processing steps should I check to improve quantification of low-intensity peaks?
 
 
Nick Shulman responded:  2026-01-20 05:44
Can you send us your Skyline document?
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.

Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

One reason that Skyline could be giving you the incorrect area for low concentration samples is that Skyline might be integrating the wrong chromatographic peak. A common problem that people have run into in Skyline with calibration curves is that when Skyline is unable to find the peak for the analyte of interest, Skyline instead integrates some other peak. In those cases, it often appears that the lower concentration samples have a much larger peak area than the higher concentration samples, but this is happening because Skyline is reporting the area of something else.

How wrong are the areas that Skyline is reporting for your low concentration samples? If they are very far off then it might be because Skyline is picking the wrong peak.

Skyline's way of computing background is probably different from the way your other software does it. You can read about Skyline's technique here:
https://skyline.ms/home/software/Skyline/wiki-page.view?name=tip_peak_calc

We might be able to give you better advice after we see your data.
If you have screenshots of other software that you are using that might be helpful for us to see especially if your other software is getting better results.
-- Nick