If you have made a change which affects chromatogram extraction, such as adding new targets, changing the settings, you usually have to tell Skyline to extract chromatograms again before you will see the effect. The usual way to do that is with the "Reimport" button at "Edit > Manage Results". Reimporting in this way will preserve any manually adjusted peak boundaries.

Skyline extracts chromatograms by summing the intensities in spectra in a narrow m/z range around the transition m/z. The width of the channel that Skyline sums across is controlled by the "Resolution" or "Mass Accuracy" setting on the "Full Scan" tab at "Settings > Transition Settings".
The setting is called "Mass Accuracy" if you have told Skyline to use centroided spectra; otherwise it is called "Resolution". In both cases, the setting controls the width of the m/z channel that Skyline sums across when extracting chromatogram intensity values.
If you click on a point along a chromatogram, Skyline will display the spectrum which contributed to that extract ion chromatogram point. The spectrum will show a highlighted region around each transition m/z showing the width of the channel that Skyline summed across.

Skyline usually tries to extract chromatograms for the entire precursor isotope envelope from the MS1 spectra. The number of isotope peaks that Skyline extracts chromatograms for is controlled by the "Isotope peaks included" setting on the "Full Scan" tab at "Settings > Transition Settings".
Skyline will never extract a chromatogram for a precursor isotope m/z whose predicted abundance is less than 1% of the most abundant isotope m/z, so, for very small molecules, it is impossible to get Skyline to extract a chromatogram for more than one precursor m/z.

When Skyline has extracted chromatograms for more than one precursor isotope, the goodness of fit (dot product) of those relative intensities to the predicted isotope distribution is one of the values which Skyline uses to decide which chromatogram peak to choose. You can see how Skyline ranked the detected peaks with the Candidate Peaks window. In Skyline 24.1 the Candidate Peaks window can be found at "View > Other Grids > Candidate Peaks" and in Skyline-daily at "View > Live Reports > Candidate Peaks".

If you have more questions it might be helpful if you could send us your Skyline document and one or more of your raw files.
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.

The Share Document dialog gives you the option of including raw files in the .zip, or you could send them to us separately.

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
-- Nick

Dear Nick and Team,

You provide outstanding support, and I truly appreciate it! Thank you for the detailed guidance and instructions. I will do my best to follow them, and without a doubt, if I encounter any issues, I’ll upload the files and would greatly appreciate your guidance.

Thank you again for your thorough explanations and the speed of your response—it’s genuinely above and beyond.

Michal

Hi,

Following up on my earlier question about mass accuracy, I would like to configure Skyline to extract signals only if their mass accuracy is within 5 ppm or less.

I am working with Orbitrap data - should I adjust any settings under the 'Library' tab in the Transition Settings menu to ensure this accuracy threshold is enforced?
Currently, with my 'full scan' settings configured for an Orbitrap instrument at a resolution of 70K at 200 m/z, I observe that some of the metabolites extracted by the software have high mass errors, occasionally reaching 7-8 ppm.

Thank you for your assistance!

It sounds like you need to adjust the value for Transition Settings > Instrument > Method Match Tolerance m/z.

The "Method match tolerance m/z" is actually for something completely different.

The Method match tolerance m/z is used to decide whether a particular MS2 spectrum should be used when extracting chromatograms from MS2 spectra.
It is also used when deciding whether the Q1 and Q3 values in an SRM chromatogram are close enough to the precursor and product m/z for the chromatogram to match a Transition in the Skyline document.

Also, the "Ion match tolerance" that you found on the "Library" is only used for annotating ions in a spectral library.

I am not sure exactly what you mean by "extract signals only if their mass accuracy is within 5ppm or less".

One thing is that for Thermo instruments, we generally recommend choosing "Centroided" for the "Mass analyzer" on the "Full Scan" tab at "Settings > Transition Settings".
The reason for this is that Thermo's software does a good job of separating signal from analytes with similar m/z values.
When you choose "Centroided" as the mass analyzer you are able to specify the mass accuracy instead of the resolution. The mass accuracy and resolution settings both control the width of the m/z channel that Skyline sums across when extracting a chromatogram point. However, with centroided data you are able to choose to make that channel narrower.
If you set the mass accuracy to 5ppm, then the chromatogram will have zero intensity values in the spectra where the centroided m/z is more than 5ppm away from the m/z of the transition.

Usually, you would not want to set the mass accuracy of the chromatogram extraction to such a low number. When the mass spectrometer has a 5ppm mass accuracy, there are still usually many spectra where the centroid's m/z ends up being outside of that tolerance because of random noise. If you set the mass accuracy of chromatogram extraction too low the chromatogram will end up being jagged as it jumps between spectra where the centroid's m/z was within the extraction window or not.
-- Nick