When Skyline is extracting chromatograms from MS2 spectra, Skyline compares the m/z's of the precursors in the document to the "isolation window target m/z" (i.e. the m/z that the mass spectrometer selected for fragmentation) value for the spectrum.

The "method match tolerance m/z" setting can be found at "Settings > Transition Settings > Instrument". In your document, the method match tolerance m/z is 0.055 (which is the default value).

When the acquisition method is "PRM", the difference between the isolation window target m/z and the monoisotopic m/z of the precursor in the Skyline document needs to be less than the method match tolerance m/z.

When the acquisition method is "DDA", the way that Skyline decides whether a spectrum matches a precursor in the document is a little more complicated because it involves Skyline calculating the predicted isotope distribution of the molecule, and using the precursor resolving power on the "Settings > Transition Settings > Instrument" tab. Basically, if the m/z that the mass spectrometer isolated overlaps with the MS1 extraction window for any of the precursor isotope transitions, then that MS2 spectrum will be used when extracting an MS2 chromatogram for that precursor.

For the peptide "VVSVLTVLHQDWLNGK" in your document, the charge 3 precursor m/z is 603.3403.
When I use ProteoWizard SeeMS.exe to look at your raw file, I see that there are some spectra where 603.67767 was isolated.
Since that difference is more than 0.055, those MS2 spectra will not be included in the extracted chromatograms.

If you go to "Settings > Transition Settings > Instrument" and change the "Method match tolerance m/z" to a larger number such as 0.5, then those spectra would be included.

The difference between 603.3403 and 603.67767 is about one third, so it looks like the mass spectrometer was told to isolate the M+1 peak instead of the monoisotopic peak.
Do you know why the mass spectrometer was told to do this? Sometimes that means that the chemical formula for the peptide was incorrectly calculated by the person creating the instrument method.

I hope this helps.

By the way, the best way to send someone a Skyline document is to use the menu item:
File > Share

The "File > Share" menu item creates a .zip file containing the Skyline document and supporting files including extracted chromatograms (.skyd), background proteome, etc.
-- Nick

Nick, thanks for your quick and detailed response, helped a lot!

"The difference between 603.3403 and 603.67767 is about one third, so it looks like the mass spectrometer was told to isolate the M+1 peak instead of the monoisotopic peak."

Yes, I did target the M+1 peak because it was of somewhat higher abundance compared to the monoisotopic peak. Guess it would have made more sense to go for the monoisotopic though (lol).

People usually only target the monoisotopic peak for fragmentation in an experiment like this. One of the reasons for this is that when you fragment a monoisotopic molecule, you only get monoisotopic fragments. When you fragment a molecule which is one Dalton heavier than the monoisotopic molecule, some of the fragments have the monoisotopic mass and some are one Dalton heavier, depending on whether the heavy atom happened to part of the fragment.

For this reason, even if the M+1 peak may have higher intensity in the MS1 spectrum, if you were to fragment that peak, you would probably observe lower signal for the fragment than if you had fragmented the monoisotopic peak instead.
-- Nick