*2.5. Metabolomics*

Besides the protein or peptide components, metabolites in mucus were also determined by UPLC-QTOF MS/MS and identified from common metabolite databases by comparing the molecular weights. Principal component analysis (PCA) provides a summary of all the observations, revealing significant differences between mucus and tissue in both positive-ion (Figure 5A) and negative-ion (Figure 5C) detection modes. Similar results were obtained by orthogonal partial least-squares-discriminant analysis (OPLS-DA) and was subsequently used to determine the most significant metabolites in mucus and tissue in both positive-ion (Figure 5B) and negative-ion (Figure 5D) MS detection modes using the variable important plot (VIP) value (>1). The R2X, R2Y, and Q2Y values in the positive ion mode were 0.884, 0.999 and 0.997, respectively, whereas their values in negative ion mode were 0.712, 0.998 and 0.993, respectively, indicating a good predictive power and goodness-of-fit of OPLS-DA plots.

A total of 16 discriminating metabolites with three subgroups were obtained (Table 1) according to FC values of mucus vs. tissue. The FCs of nine metabolites including L-Glutamate, Succinylacetone, Linoleyl linolenate, Uridine, L-Proline, Inosine, Hypoxanthine, L-Valine and Guanosine are smaller than 1, representing a lower concentration in mucus than that in tissue. Five metabolites including 4-Hydroxy-L-proline, Citrulline, L-Leucine, 3-(Phosphoacylase mido)-L-alanine and L-Threonine have FC values ≈ 1, indicating similar concentrations between the two groups. The metabolite Tryptamine, a derivative of Tryptophan, is a potential neurotransmitter or neurotransmodulator, and displayed the largest FC value of 7.8. This value was significantly higher than all other values and indicates elevated enrichment in mucus over that of all other metabolites. This feature is of particular interest to us. A further KEGG scanning of Tryptamine shows that there are 16 proteins identified from proteomics and 26 mRNAs from transcriptomics (Figure 5E) in tissue homogenate. Only 1 'mucus-enriched' protein identified was matched to the downstream region of the 'Tryptophan metabolism' pathway. This indicates a strong Tryptophan/Tryptamine metabolic system in the cytosol, but not in mucus, allowing for Tryptamine synthesis or accumulation.

**Figure 5.** *Cont*.

**Figure 5.** Plots of multivariate statistical analysis of all experimental groups in electrospray ionization (ESI) positive and negative-ion MS detection modes. The difference in substances between the two groups was screened by a variable importance plot (VIP). (**A**) Principal component analysis-X variogram (PCA-X) (+) and (**B**) Orthogonal partial least-squares-discriminant analysis (OPLS-DA) (+) scores plot of the mucus and tissue groups in the ESI positive-ion mode. Asterisk (\*) indicates multiplication sign. (**C**) PCA-X (−) and (**D**) OPLS-DA (−) scores plot of the mucus and tissue groups in the ESI negative ion mode. The red plots represent the data from the secreted mucus, whereas the green plots represent data from the tissue homogenate. (**E**) The 'Tryptophan metabolism' pathway from KEGG PATHWAY database where the mRNAs from the transcriptomics of jellyfish tissue are colored by red, while the light blue and white boxes represent the background genes annotated or not in the KEGG PATHWAY database. Meanwhile, the metabolites 'Tryptophan' and 'Trytamine' are highlighted by blue.



Note: FC values were obtained by comparing the mean concentration of each metabolite in mucus with that in tissue; FC value > 1 indicates a higher concentration in mucus, while the value < 1 indicates a lower concentration in mucus (*n* = 10). Metabolites analyzed based on MS/MS chromatograms.
