Computational protocol: Effect of masticatory stimulation on the quantity and quality of saliva and the salivary metabolomic profile

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Protocol publication

[…] Raw data were analyzed using MasterHands (version, Keio University, Yamagata, Japan), which detected all possible peaks, eliminated noise and redundant features, and generated the aligned data matrix [, ]. Metabolite identification was conducted by matching the m/z values and the normalized migration times with our standard compounds, and the concentrations of individual metabolites were calculated using both internal and external standards [].Principal component analyses (PCA) were conducted to assess the overall diversity in the metabolomic profiles. The first and second PC (PC1 and PC2) were individually analyzed by subject characteristics. Using two-dimensional PCA, Takeda et al. reported that salivary metabolites from samples taken at rest differ from those obtained after stimulation of saliva production by acid []. Analogous to these analyses, we assessed the differences between saliva taken at rest and saliva taken during stimulation by mastication. The Wilcoxon matched pairs test, Spearman correlation, and Wilcoxon test were used for the analyses of sampling methods and the other characteristics, respectively. The Kruskal-Wallis test was used to assess the age-dependent differences. P-values for metabolites were corrected using a false discovery rate (FDR) to account for multiple testing []. Metabolite data showing correlated P-values that were less than 0.05 were transferred to pathway analysis using MetaCore (Thomson Reuters Inc., Carlsbad, CA). The geometric mean of fold change (FC) of the concentration of stimulated saliva divided by the concentration of unstimulated saliva was calculated among samples. Maximum FC and half of the minimum FC among the 55 subjects were substituted for the undetected cases in unstimulated and stimulated peaks, respectively.These data analyses and visualizations were conducted XLStat (version 2014.1.09, XLStat, Paris, France), R-software (2.14.0, R Foundation for Statistical Computing, Vienna, Austria,, and GraphPad Prism (version 5.0.2 GraphPad Software Inc., San Diego, CA). […]

Pipeline specifications

Software tools MasterHands, MetaCore
Application MS-based untargeted metabolomics
Chemicals Cysteine, Glycine, Urea