Review for "LAESI mass spectrometry imaging as a tool to differentiate the root metabolome of native and range-expanding plant species"

Completed on 21 Jun 2018 by Scott Edmunds .

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The authors used LAESI MS imaging technique as a tool to differentiate the root metabolome and applied to compare native vs range-expanding plant species. This is an interesting idea and application. It has a potential to be publishable but will need a major revision to make it more meaningful.

Most importantly, biological meaning is almost completely missing in the current manuscript. It is not surprising to see some differences in the metabolome of range-expanding plant species when compared to native ones. Question is what the difference tells us. In regards to this, they did not identify any of the metabolites that show significant changes in the volcano plots. They are simply showing the m/z values. Those metabolites should be identified in combination of MS/MS and accurate mass analysis, as well as in-parallel GC-or LC-MS analysis if necessary, so that the meaningful biological interpretation can be drawn.

Here are some other minor comments:

1. P1, line 19: 'laser-assisted ...', -> 'laser-ablation'.

2. P2, line 43-44: 'ionize the sample to form ions ...', rephrase. 'ion' is redundant.

3. P2, line 44-46: "Different ionization methods exist for MSI, however many of them require artificially altering the biochemical status of the system under study, for example by the use of a matrix, and are mainly operated under vacuum.". I do not understand what it means. In typical MSI, the tissues are processed in a way the metabolism is quenched, typically free-drying. Biochemical status is not expected to be changing if the sample prep is done right. P

4. P4, line 94-96: "Since all the 105 LAESI ablation spots for a single replicate were not present on the root sample, mass spectra arising from only 50 spots per replicate, that were visibly present on the root sample were selected manually." I couldn't understand this sentence until I completely read the manuscript. It should be rephrased for the readers to easily understand as they are reading.

5. Figure1. They claim 'clear distinction in mass spectra between the two plant genera' (line 107), but it is hard to tell as. They should do something to make the difference clear, such as labeling some clearly distinct peaks with different colors.

6. They 'manually' selected some spectra from the data they acquired. What is the guideline for such selection? It needs to be clarified.

7. They used 'imaging' in the title but there is no MS imaging in the paper. They should drop 'imaging' from the title. It is misleading.

8. P8, 214: ' First, square root transformation was applied to overcome the dependency of variance on the mean.', what is that mean?