|Small Molecule MRM extraction with isomers||wbarshop||2018-08-13|
Hello Skyline team,
We optimized collision energies via direct infusion, and started by utilizing the observable fragments and their empirical masses. Of course, when we switch from isoform to isoform for this process on our QQQ, the masses reported back vary a little, even if the fragments being monitored are the same.
My suspicion [based on very little] is that when we have multiple small molecules where transitions exist with the same (or very similar) m/z values for both the precursors and fragments, the matching behavior of Skyline (via the "Method match tolerance m/z" setting) may be getting tripped up and leaving out data.
I have included a simplified Skyline document with four analytes. All share the same precursor m/z, several of them share all transitions (often within the limit of the method match tolerance m/z setting). In this case, you can see the "Deoxycholic acid" analyte loses its transition extraction (despite skyline having the exact m/z values stated by the instrument method-- though I know some values like monoisotopic m/z and average m/z seem to be float representations and not precise).
In this Skyline file, you'll notice that Deoxycholic acid fails to extract its fragment ions. If you modify the small molecule and remove the explicit retention time, you'll see that we extract the peaks from the "3b12a" molecule, as they are within the method match tolerance.
Even, though, if I drop the method match tolerance to be very narrow, I cannot seem to recover the extraction of the proper MRM data for "Deoxycholic Acid."
I am included here the simplified skyline file, along with the same RAW file which is imported into the sky file. Please let me know if you need any additional information or data, I will be happy to provide.