Review for 10.1186/s12876-023-02803-6 (2023)

Title: Changes in the salivary metabolome in patients with chronic erosive gastritis
Version: 2 Date: 10 Oct 2022
Reviewer's report:
Dr. Shaowei Liu’s study analyzed the salivary metabolites using LC-QTOFMS to find the
metabolites showing discrimination abilities for chronic erosive gastritis. The topic itself is
important, however, there are many sever problems in this manuscript.
The most significant problem is the reproduciliby was not confirmed. The linearity between
mass spectrometry signal and actual metabolite contents were also not evaluated. The authors
did not mixed internal standards when they process saliva samples. Thefore, the authors did not
evaluate the recovery rate of each metabolites. The dynamic range of each metabolites were
also not evaluated. At least, the authors must evaluate these fundamental factors before the data
analyses. I understand the authers normalized the data by total peak intensity in data processing
procesure. This process might be expected to eliminate the fluctuation of sensitivity of each
measurement of MS. However, total intensity include background noise and the normalization
using the total intensity is not appropriate with the author’s measurement protocols.
At least, the authors must the reproducibility and linearity using standard mixture. Subsequently,
the standard mixture shoud be spiked into each saliva samples, and the recovery rate should be
evaluated. Internal standards also should be added when saliva samples is mixed with methanol/
acetonitrile/aqueous solution. The signals should be normalized the the internal standards.
The statistical analyses were completely wrong. Whey Student’s t-test was used? Mann-Whitney
test is appropriate instead. To control the alpha error considering multiple indepenent test, p-
values must be corrected by several methods, e.g. the use of Bonferonni correction.
The 7-fold of cross-validation of OPLS-DA have shown overfitting in many cases. The authors
should read review papers of PLS-DA. At least double-cross validation and also indepenend
valdation is necessary.
The cutoff of VIP > 1 is not no theoretial. Based on the distribution of VIP values in the authors’
data, the appropriate cutoff should be defined.
Anyway, the current data analyses is meaningless. The current data did not conclude the
metabolite difference is attributed to the biological difference.
Minor issues:
2.2.Sample collection and preparation.
- The required protocols before the sample collection, e.g. the fasting duration, the use of tabacco,
and excericse, was not described. These factors affects salivary metabolomics.
- Centrifuge condition, “14000 R /min” is not appropriate for scientific papers. “x g” should be
used instead.
- The first sentence of the second paragraph is not collect English. Do not use “add”.
- Explain abbreviations at the first usage, e.g. UHPLC.
2.3. LC-MS/MS Anlysis
- Title needs space between number and title.
- “quadropole time-of-flight” is not an instrument. This is just an interface.
2.4 Raw data processing and statistical analysis
- There was no description for the detail of each data processing. How do readers reproduce
these procedures?
- No used software description here.
3.2. LC-MS method validation.
- XCMS software (SIMCA-P 14.1)… These maybe mistake. XCMS is freely available R-based
software. SIMCA-P might be used for the PLS-DA. Also, such description should be written in
the Material and Method section.
- The correlation of QC is not enough to validate the LC-MS data. Why 0.9 is the appropriate
cutoff? All these description looks not scientific ones.
I stop the reading. The following parts are meaningless.
More scientific descripton is necessary for all parts. ROC should be written with p-values.
Even though I’m not English native, I found many English problems. The cited reference is not
appropriate, e.g. why the reference of Mu et al 2019 is appropriate for explaining metabonomics?