*4.5. Proteomics*

### 4.5.1. Protein Sample Preparation

The proteins of jellyfish mucus and tissue homogenate were precipitated with four times the volume of trichloroacetic acid (TCA) at 4 ◦C overnight. The sediment was collected after centrifugation at 15,000× *g* for 15 min at 4 ◦C, further treated with a pre-cooled acetone solution for 30 min, and re-centrifuged at 15,000× *g* for 15 min at 4 ◦C. After freeze-drying, the precipitates were dissolved in the lysis buffer (8.0 M Urea, 1× Protease inhibitor, 100 mM Tris-HCl, pH 8.0) overnight. Subsequently, the undissolved debris was removed by centrifugation at 15,000× *g* for 15 min at 4 ◦C, whereas the supernatant was stored at −80 ◦C for further use.

After mass quantification of the pre-treated samples, 60 μg mucus or tissue homogenate were mixed with 5 μL 1 M dithiothreitol (DTT) at 37 ◦C for 1 h, and reacted with 20 μL 1 M indoleacetic acid (IAA) in the dark at room temperature for 1 h. After that, samples were collected by centrifugation in ultrafiltration tubes and sequentially rinsed three times with 100 μL UA solution (8 M urea, 100 mM Tris-HCl, pH 8.0) and 100 μL 50 mM NH4HCO3, respectively. Finally, the collected samples were digested with Trypsin (protein:trypsin = 50:1) at 37 ◦C for 12–16 h, and the digested proteins were lyophilized and stored at −80 ◦C for further use.
