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Messages - ThorstenG

MS-DIAL / Re: Verifying MS1 Identification Accuracy

If you only have MS1 information (accurate mass in a scan and retention time), you will have difficulties evaluating your annotations further, without more analysis.

Based on your described information (MS1, RT, Name or Structure) alone;
- First, you can use the predicted elemental composition based on accurate mass and isotope pattern. It might disagree with your annotation by the library (your plant metabolite). But to be honest, that doesn't get you very far and as far I know is implemented into mzMine already.
- Second, there are some tools/ways to estimate retention times, which might help you exclude your annotation.
You can also make a crude estimate yourself using logP values and molecular surface (e.g. lipophilic cholesterol shouldn't elute early in reverse-phase chromatography on a C18 column). However, you will need to understand your method and elution behaviours well before making any such conclusions and they usually are crude estimates. 
- You can also search for isomers of your plant metabolite. Maybe there is a common animal metabolite known to be an isomer to your plant metabolite.

So, what else can you do?
  - One option is a re-injection of your sample (or a pooled QC) which shows your plant metabolite, and use a targeted MS2 acquisition list to obtain fragmentation spectra (MS2). Then you can try to match/dismatch it to spectral libraries.
  - With an MS2 in hand you might even attempt some structure elucidation (SIRUS, etc.)
  - If MS2 is not an option (you might run on a low-resolution single quad MS), you could get a reference standard for those compounds. However, that seems like a lot of (financial) effort to evaluate possible annotations at this level. If that's not an issue, because you already have a standard or it's cheap, run it and compare your retention time information.

As a further note, you might find plant metabolites in some animal tissue, e.g. through diet or because they are animal and plant metabolites.
And remember to clearly communicate your annotation level for all metabolites, even if they fit nicely into your study (animal metabolites). Just because something fits into your hypothesis/tissue/study doesn't mean its also correctly annotated, especially on MS1.

Good luck with your study and welcome to the frustrating but fascinating world of metabolomics annotation. ;)
Compound identification / Re: Duplicate annotations from Compound Discoverer
Hi djb17 and AMO,
This thread is already a bit older, but I thought an answer might still be helpful for others in the future.
Duplicates in CompoundDiscoverer are very common and originate from a multitude of reasons.

If duplicate compounds have near identical Mass and Retention Time, your Compound Grouping settings might be set too strictly. Chromatographic shifts can be corrected by alignment, but even after that, the same compound might shift a few seconds during your sequence. Hence, you need to allow CD enough room to group multiple detections. The same applies to mass accuracy and the resulting MW (mz). I cannot suggest default values since it depends on your method.

In addition, CD can have difficulties with adducts, finding e.g. the M+H and the M+NH4, then reporting both as separate compounds. I would suggest adding suggested/expected ions to the Detect Compounds node.

Once the "processing duplicates" are addressed, you get to the more complicated but essential issue of duplicate annotations.
You mentioned you see the "molecular weight are the same, but some (or all) of the retention times are different".
That indicates you are annotating based on exact mass. Many compounds, such as isomers, might have identical mass but different retention times. Significant changes (method dependent) in retention time indicate actual different compounds. CompoundDiscoverer(or any software for the matter) just cannot tell these compounds apart by mass alone.

Possible solutions to elucidate which peak is the "true" compound, are MSMS fragmentation spectra and reference standards, run on your instrument.

In general, it's important to double-guess the annotation and compounds CD provides to you. Emma Schymanski et al. summarised the annotation levels in  "Identifying Small Molecules via High Resolution Mass Spectrometry: Communicating Confidence" very well. Have a close look and ask yourself where each of the Compounds in your CD results stands on the scale.

Finally, even when you know more about the Compounds detected and their annotation, you should still filter through your CD results. Peak quality parameters, such as S/N, precision over multiple injections, annotation sources, MS match scores and gap fill status, give you numeric parameters that help you filter your results, either directly in CD or in software after export (R, Matlab, Excel).

In any case, good question and good luck with your analysis, where ever you already are at this time point. :)