Extracting precursor intensities from MS2 targeted scan

Extracting precursor intensities from MS2 targeted scan kentsisresearchgroup  2018-12-20

Dear Skyline community,

we used a Thermo Fusion Orbitrap to collect targeted MS2 of peptides, with an isolation window including two isotopologues. We are now trying to use Skyline to quantify these peptides based on their precursor intensities, extracted from these MS2 scans (the precursors are indeed detected, based on inspection of the spectra). However, for most peptides the light isotopologue is not extracted at all by Skyline, despite being in the targets list.

Can this depend on the scan header? Does the software filter spectra in any way, for example based on the nominal target mass in the header? Is there anyway to force Skyline to use all scans covering a mass range containing both isotopologues?

Thank you very much in advance for your help,

Paolo Cifani
Kentsis Research Group
Memorial Sloan Kettering Cancer Center

Nick Shulman responded:  2018-12-20
The way that Skyline decides which MS2 scans go with which precursors is based on the settings that you have specified at:
Settings > Transition Settings > Full Scan

If the "MS/MS filtering Acquisition Method" is "Targeted", then any particular MS2 scan will only be associated with the precursor whose monoisotopic mz is closest to what was isolated in the scan.

Since your scans are supposed to be assigned to both the light and heavy precursors, you should specify that the acquisition method is "DIA". When it is DIA, each scan will be assigned to all of the precursors whose monoisotopic m/z is within the scan's precursor isolation window.
You should also set the "Isolation Scheme" to the one called "Results Only", which will tell Skyline to assume that the isolation window specified in the .raw file is correct.

This will work so long as your isolation windows do not overlap with each other. If your isolation windows overlap with each other, you will find that certain chromatograms have points from scans with different isolations, and so the measured intensity fluctuate between scans depending on how much of isotope envelope was isolated.

If something like that is happening, there might not be anything that we can do about it, but you can try sending us your Skyline document and raw files.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.

You can upload that .zip file and your .raw files here:
-- Nick
kentsisresearchgroup responded:  2018-12-21
Hi Nick.
Thank you very much. Changing the settings for acquisition methods and isolation scheme seem to be working for our purpose.

Thank you and happy holidays,