Hey there. This is my first time posting a question, and I've gotten bits and pieces of an answer from other posts, but here's the gist:

The lowdown

I'm working with metabolites (so, molecule settings), and with .RAW files from a Thermo Orbitrap Fusion Lumos. We're running it in targeted mode, so MS2s are only collected when the precursor shows up. We're quantifying based on MS1 ratios to heavy (13C6), and verifying the location and identity of the EIC based on MS2 products. The fragments in question generally seem to be in the files--I have attached a screenshot of MZmine, which shows the rt locations of several MS2 scans and the expected product ions for pantothenic acid.

Skyline, however, does not show these ions. This is not always the case. When a really concentrated standard is run (>100x my highest standard curve point; not included in the attached doc), Skyline picks up on some of the transitions; however, I would assume that since the peaks in a "normal" sample still can appear at e4-level intensity, I am suspicious that there is an over-restrictive baseline calculation algorithm hidden somewhere I just haven't found.

notes

• Transition list items denoted with a "1" are the focus. Those denoted "0" are those that do not have the 13C label, and cannot be quantified as a ratio.
• I do still have an unresolved issue that may or may not matter. In Settings>Transition Settings>Filter>Fragment adducts, I cannot change the product adducts to [M+]. This doesn't seem to matter as the transitions themselves (right click on transtion, Modify...>Ion chemical formula) all show [M+] as they should. If I try to adjust the global setting; however, it reverts back to [M-] as soon as I close the window.
• I have been reading through this post and this post. I tried a couple of things, including setting my m/z match tolerance really high (0.5). No dice.