Sample collection and ethics statement
Juvenile E. sinensis (approximately 7.5 g) with the same growth conditions were collected from the Aquatic Animal Germplasm Resource Station of Shanghai Ocean University (Shanghai, China). They were cultured in a circulating water system for seven days to adapt to the environment. Then, the E. sinensis individuals 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 E. sinensis individuals were reared in the circulating water system, named “Crab Dragon Palace” to maintain consistent culture environment. The water temperature was maintained at 26 ℃ ± 2 ℃, and the three groups were fed with abundant food at 9:00 and 16:00 every day, respectively. When the E. sinensis grew to the prestage of molting, their hepatopancreas tissues were collected. Three biological replicateswere collected for each group. Then, the tissues were quickly frozen in liquid nitrogen and stored in a −80 ℃ refrigerator. The study was approved by the Institutional Animal Care and Use Committee of Shanghai Ocean University (Shanghai, China). Sampling procedures complied with the guidelines of the Institutional Animal Care and Use Committee on the care and use of animals for scientific purposes.
Protein extraction and quality control
The collected hepatopancreatic tissues were taken 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 on an ice bath for 30 min. Centrifugation was performed at 14 000 g for 10 min at 4 °Cand the supernatant was collected. Protein concentration was measured with a Pierce BCA protein assay kit in accordance with instructions (Thermo fisher, USA), and protein quality was evaluated through SDS-PAGE gel electrophoresis.
Protein alkylation, trypsin enzymatic hydrolysis, and TMT tagging
The proteins were alkylated in accordance with Randall’s protocol (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 peptides were desalted and lyophilized. A total of 100 µg protein was taken from each sample for TMT labeling by using the 10-plex TMT reagent (Thermo fisher, Art.No.90111) according to the manufacturer’s instructions. Three biological replicates were sampled for each group. The labeling steps were as follows: First, 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 Xcalibur 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 data-dependent acquisition (DDA).
Proteome DiscovererTM software 2.4 was used to search the Eubrachyura Uniprot database and our assembled reference transcriptome (15) to identify and quantify proteins. The MS/MS search criteria were as follows: Mass tolerance of 20 ppm for MS and 0.02 Da for MS/MS Tolorance, trypsin as the enzyme with two missed cleavage allowed, carbamido methylation of cysteine and the TMT of N- terminus and lysine side chains of peptides as fixed modification, and methionine oxidation as dynamic modifications, respectively. False discovery rate (FDR) of peptide identification was set as P ≤ 0.01. A minimum of one unique peptide identification was used to support protein identification. Proteins with fold change (FC) < 0.667, or FC >1.5, and P < 0.05 for the FDR were considered as differentially expressed proteins. Pairwise comparison was conducted between every two groups. Differentially expressed proteins were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis by using the software implemented in Majorbio I-Sanger Cloud Platform with corrected P < 0.05.