Inquiry about precursor mass consideration and collision energy combination in ms2query

[Javi-Rop]

Hello

First of all, thank you for developing this fantastic tool; it has been tremendously helpful in my workflow. I have a couple of questions regarding the annotation process and collision energy settings:

• Precursor Mass Consideration
  I’ve noticed that sometimes the precursor mass measured on my instrument differs significantly from the mass reported in the tentative molecular annotation. This seems to indicate that the annotated molecule may be entirely different from what I actually have. Therefore, I’m wondering:

What is the advantage of generating a tentative molecular annotation without considering the precursor mass?
Is there an option to narrow or constrain the precursor mass error so that ms2query only searches within a defined mass window?

• Combining Collision Energies
  I typically acquire data at 10, 20, and 30 eV collision energies. I’d love to hear your opinion on whether it is advisable to combine multiple collision energies in a single run of ms2query or if it’s generally more reliable to process each energy separately.

Thank you in advance for your time and for any insights or recommendations you can provide. I truly appreciate all the work you do in maintaining and improving ms2query.

Kind regards

[niekdejonge]

Hi @Javi-Rop,

Great to hear that MS2Query has been useful for your research!
Regarding precursor mz: MS2Query is designed to not only return exact matches, but also "analogues", which means molecules that are not exactly the molecule you are looking for, but molecules that are chemically similar to the molecule you detected. The reason why we do this is that for many metabolites there is no reference standard available. This would mean that it is impossible to get an exact match for these metabolites. By giving a close analogue instead it can still help you to get an idea if this could be of a molecule of interest for you. E.g. if you are trying to find potential drug breakdown products it can be helpful to find that a metabolite is an analogue of the drug, helping you prioritize these metabolites for instance for annotation by NMR. Another option is using the mass difference between the found metabolite to do annotation propagation, so using the analogue and mass difference to predict what the actual metabolite could be, however this can be challenging and will require confirmation with a standard afterwards.

If these "analogues" are of no interest to you, the easiest way is to just filter out all hits with a mass difference from the csv file, this will result in less results, but all are likely exact matches. It is also important to only consider MS2Query hits that have a high enough score. The higher the more likely it is a good match. But in general I would not take scores below 0.6 or 0.7 as good matches.

Regarding collision energy. I have not experimented myself with the effect of combining collision energies into one merged spectrum before running MS2Query. MS2Query was designed and trained to match spectra of a single collision energy. Therefore I would advice to analyze them separately and pick the hit with the highest MS2Query score, ideally the 3 collision energies give the same or similar results. Alternatively you could pick the collision energy that fragmented best before running MS2Query. We will in the future likely release a version of MS2Query that has the option to combine multiple collision energies as input for annotation to further improve reliability, but this will still take quite some time to develop.

Hope that helps.
Best,
Niek