Ghost peak in Skylin

Ghost peak in Skylin ychiu2  2022-10-07 12:02

Hello Skyline team,
I have been using Skyline to do the quantification for small molecule and recently we found a ghost peak integration issue. In Skyline, you can see that the software integrate a peak at m/z 1055.5057, but when I plot m/z 1055 in Freestyle (Thermo software), no peak was found. I really want to know if there is a way to fix this as we use Skyline heavily for quantification. If the peak integration was not good, then the data validation will be very difficult. I have attached the skyline file and two graphs demonstrating the peak
Thank you

Nick Shulman responded:  2022-10-07 14:17
Can you send us your .raw file ("20220923_E_1_ControlOyster_0ng.raw")?
If that file is less than 50MB you can attach it to this support request. You can upload larger files here:

When Skyline is extracting chromatograms, Skyline looks at each spectrum and sums up the intensities that are observed in a m/z range around the target transition's m/z. If you are seeing a peak in the Skyline chromatogram that you are not seeing in the chromatogram extracted by your Thermo software, then it is probably because Skyline is summing intensities across a wider m/z window than your other software.

The width of the m/z channel that Skyline sums across is controlled by the "Resolving Power" or "Mass Accuracy" setting that you have at "Settings > Transition Settings > Full Scan".
For the chromatogram in your screenshot, that's going to be controlled by the Resolving Power that you have specified in the "MS1 filtering" section. Your Resolving Power is set to 60,000, which should result in summing across a narrow channel (also, the "Use high-selectivity extraction" that you have checked on that page also means that Skyline will use a window which is half as wide as it would otherwise.

If you want to see how wide of a channel Skyline is summing across, you can click on any point along the chromatogram graph.
When you click on the chromatogram graph, Skyline brings up the "Full Scan" graph which will have shaded rectangles on it which show what m/z windows Skyline was summing across.
-- Nick
ychiu2 responded:  2022-10-10 07:19
Hi there,
I have uploaded the file into the folder as the raw file is too big. Thank you!
Nick Shulman responded:  2022-10-10 07:50
If you click on a point along the chromatogram, Skyline will show you a graph of the spectrum and also indicate how wide of a m/z channel Skyline was summing across.
I see in the attached picture "SpectrumWithOriginalSettings.png" that the signal that Skyline is seeing on that big chromatogram peak is mostly located on the right side of the extraction window. That means that it would be possible to cause that signal to not appear in the chromatogram if you go to "Settings > Transition Settings > Full Scan" and change the settings to be more exclusive.

If I change the mass analyzer to "Centroided" and the mass accuracy to "5ppm" ("Centroided5ppmSettings.png"), then, when I do "Edit > Manage Results > Reimport", the big peak disappears from the chromatogram ("Centroided5ppmChromatogram.png").

What settings did you use with the Thermo software when you told it to extract the chromatogram for you?
-- Nick