The problem is that the resolution that you have specified at:
Settings > Transition Settings > Full Scan > MS/MS filtering
is too high, and so the m/z channel that Skyline is summing across is so narrow that it does not include any of the profile data that Skyline sees.

If you click on a point along the chromatogram, Skyline will show you the spectrum which contributed to the point (see attached picture). The shaded purple region represents the m/z channel that Skyline summed across. The vertical lines under the yellow shaded region represent the m/z and intensity values in the spectrum. When Skyline is calculating the chromatogram intensity, Skyline does not do any sort of interpolation between those m/z's. Skyline just sums up all of the peak heights that appear within the shaded purple rectangle. In this case, that purple rectangle is perfectly centered between a pair of points, so the chromatogram intensity is zero.

For Orbitrap data, we usually recommend that you use "Centroided" as the "Precursor mass analyzer". When "Centroided" is chosen, Skyline will use Thermo's centroiding algorithm to find the center of that peak and the chromatogram intensity will end up being the area under the peak that you see there.
Alternatively, you could change the resolution to a smaller number (it is currently set to 60,000) and Skyline will sum across a wider m/z window when extracting chromatogram points.

By the way, this seems to be a DDA experiment. It looks like the mass spectrometer was looking at the MS1 spectra and deciding what to fragment based on what it saw. For this reason, your chromatograms have long straight lines which represent regions of time where the mass spectrometer happened not to collect any MS2 spectra which matched your precursor. If you are going to use your MS2 chromatograms for quantification we would recommend that you change your instrument method so that you are telling the mass spectrometer when to collect MS2 spectra for your precursors (which would make it a PRM method instead of DDA).
-- Nick

Hi Nick,

Excellent, changing the resolution worked. I honestly did not think to change the m/z resolution because I was focused on the loss of the low intensity peaks, and suspected a filter somewhere based on peak intensity.

I am curious about using "Centroided" as the "Precursor mass analyzer". What is SkyLine doing when we select "Orbitrap"? This question is entirely based on curiosity and wanting to learn more.

We are not using the MS2 peaks for quantification, they are just to confirm our identifications. Moving forward we may use a combination of a targeted list to fragment and open-ended fragmentation based on 'top-five peaks', but we are still testing that out.

Thanks, for your help.
Krista

Skyline asks you the mass analyzer and resolution in order to decide how wide of a m/z channel to sum across. With a TOF mass spectrometer, you only need to tell Skyline the resolution, because that resolution applies for all m/z values in the scanning range. For a Orbitrap or a FT-ICR, you have to specify not only the resolution, but also the m/z value at which that resolution applies. Then, when Skyline wants to know the resolution at some other m/z, Skyline applies a formula to convert. The conversion formula is different for Orbitrap and FT-ICR. One of them involves squaring the m/z value, and the other involves raising the m/z to a different power.

It is ok to lie to Skyline about the mass analyzer or resolution if that causes Skyline to choose better m/z extraction widths. We added the checkbox "Use high-selectivity extraction" for people who did not feel comfortable lying to Skyline, but that checkbox has the same effect as specifying a resolution which is twice as selective.

When you choose "Centroided", Skyline asks the mass spectrometer vendor's software to convert the broad profile peaks as shown in my screenshot into a single vertical spike like shown in the screenshots in your PowerPoint. With centroided data, Skyline does not need to know how broad peaks are expected to be, since the centroiding algorithm will collapse the peak into a single point. Skyline just needs to know what the mass accuracy is, so any points outside of that accuracy window can be assumed to have not come from the analyte of interest.

For Thermo data, we recommend using "Centroided" instead of any of the other mass analyzers because Thermo's centroiding algorithm is better at looking at profile data and figuring out the center of mass and area of an observed peak, compared to Skyline's way of just summing up the numbers from the profile data which fall in the m/z range.
-- Nick

Hi Nick,
This is all extremely helpful, particularly your comment about 'lying to SkyLine'. Thank you for taking the time to so fully answer my questions.
Krista