Orbitrap FSddMS2 NCE

[gpblack]

What is everyone using for NCE for a “generic” NTA FSddMS2 run? I’m running 35, but not seeing a lot of fragmentation and contemplating if I need to increase this, or if the lack of fragments is a function of the types of compounds that are in my samples.

[Ben Place]

For me, it depends on my specific goal for the NTA run. If I want to generic reference spectra for specific compounds, I run my samples in three different NCE levels: 15, 30, 45 (individually). I’ve not found much of an issue with 30-35 for fragments.
For doing more broad NTA type runs, I run my QE in Stepped NCE levels, with 15, 30, 45 being the steps to get an average spectrum across the three levels.

For PFAS specifically, I’ve found NCE 45 tends to produce 0 – 1 fragments on the low end (m/z 80 – 99). NCE 15 tends to produce only the pseudomolecular ion, and NCE 30 tends to give me a handful of ions with a pretty big pseudomolecular ion.

[SLN-chem]

My generic methods have stepped NCE at 15, 30, 45 or 20, 40, 60.

[mnewmeyer]

My generic method has stepped NCE at 30, 65, and 100. We’re running on a Q Exactive HF which uses an m/z of 500 for normalization. The degree of fragmentation will totally depend on the individual compounds; with this setup, I still see occasional compounds with only a precursor ion peak.

It may also help in your decision-making to consider what, if any, outside libraries for spectra searching you may be using. Incorporating the stepped energies can add some additional complexity since it’s a composite. mzCloud is supposed to be able to generate a composite spectrum to match, as closely as possible, your experimental spectrum. I have had some challenges, though, comparing my spectrum to spectra found on MoNA or Massbank where a single energy was used and it’s not clear if the spectrum was generated with absolute or normalized energies.

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