Sample collection and ethics statement
Juvenile crabs (approximately 7.5 g) with similar growth conditions were collected from the aquatic animal germplasm resource station of Shanghai Ocean University. They were fed in a circulating water system for 7 days to adapt to the environment. Then, the crabs were randomly divided into three groups and fed as follows: Group A was fed with a mixed diet of S. quadrita and E. canadensis, group B was fed with E. canadensis only, and group C was fed with S. quadrita only. All the crabs were reared in the “Crab Dragon Palace” in the same environment. The water temperature was maintained at 26 ℃ ± 2 ℃, and the three groups were fed with the same amount of food at 9:00 every day. When the crabs grew to the early stage of molting, their hepatopancreas tissues were collected. Three biological repeats were set for each group. Then, the tissues were quickly frozen in liquid nitrogen and stored in a −80 ℃ refrigerator. The whole process follows the institutional animal care and use committee of Shanghai Ocean University (Shanghai, China).
Protein extraction and quality control
The collected hepatopancreatic tissues were removed from −80 °C refrigerator and homogenized. Approximately 50 mg of minced tissue was mixed with 500 µl of RIPA lysate (PMSF was added before use). Subsequently, the homogenate was incubated in an ice bath for 30 min. Centrifugation was performed at 14 000 g for 10 min at 4 °C. The supernatant was collected in a new tube. Protein concentration was measured with a Pierce BCA protein assay kit in accordance with instructions (Thermofisher, USA), and protein quality was tested through SDS-PAGE gel electrophoresis.
Protein alkylation, trypsin enzymatic hydrolysis, and TMT tagging
The proteins were alkylated in accordance with Randall (13), and the filter-aided proteome preparation method was used for protease hydrolysis (14). The trypsin enzyme was added on the basis of the ratio of protein: enzyme = 40:1. The mixture was placed at 37 ℃ overnight. Then, the peptide segment was desalted and lyophilized. A total of 100 µg of protein was taken from each sample for TMT labeling. The labeling steps are as follows: First, the temperature of the TMT reagent was allowed to recover to room temperature. Then, acetonitrile was added to the sample, and the sample was centrifuged at low speed with a vortex. Second, the sample was mixed with TMT reagent, incubated at room temperature for 2 h, and then mixed with hydroxylamine. The mixture was reacted at room temperature for 15 min. Finally, the same amount of labeled substances was mixed in a tube and drained with a vacuum concentrator.
HPLC fractionation and LC–MS/MS analysis
Polypeptide samples were redissolved with UPLC loading buffer, and a reverse phase C18 column was used to separate the high pH liquid phase. A total of 20 fractions were collected and merged into 10 fractions in accordance with peak type and time. After vacuum centrifugation and concentration, the mass spectrometry sample was dissolved with the loading buffer solution for mass spectrometry. The mass spectrometry conditions were as follows: The data acquisition software was Thermo Xcalipur 4.0 (Thermo, USA). The chromatographic instrument was Easy NLC 1200 (Thermo, USA), and the mass spectrometer was Q_Exactive HF-X (Thermo, USA). The chromatographic separation time was 120 min, the flow rate was 300 nl/min, the scanning range of MS was 350–1300 m/z, and the acquisition mode was DDA.
ProteomeDiscovererTM software 2.4 was used to search NCBI and the Uniprot database to identify and quantify proteins. Proteins with fold change (FC) < 0.667, FC >1.5, and P < 0.05 were considered as differentially expressed proteins. Differentially expressed proteins were subjected to GO and KEGG enrichment analysis by using the software implemented in Majorbio I-Sanger Cloud Platform with corrected P < 0.05.