Eksigent ultra 2d

The proteins of the samples were separated by gel electrophoresis, the samples were cut into 1 mm particles, and the peptides were obtained by enzymatic hydrolysis with 0.01μg/μL trypsin, and then frozen and drained. After being redissolved in mobile phase A (2% ACN, 0.1% FA), the peptide samples were centrifuged for 10 min at 20,000 g, and then were injected. Liquid phase separation was performed by a nano liter liquid chromatograph (Eksigent Ultra 2D, SCIEX, Framingham, MA, United States). After separation, the peptides were transferred to ESI tandem mass spectrometer (Triple TOF 5600, SCIEX, Framingham, MA, United States). Scanning was set as high sensitivity mode. Fragmentation Energy selected “RollingCollision Energy”. The analyzed data were searched against the protein database of Tilapia for peptide matching.

Surface proteins of A. flavus were obtained according to the method reported previously with slight modifications [21 ]. In brief, 1 g of fresh mycelia were washed several times with deionized water, suspended in 5 mL of 0.156 M phosphate-buffered saline (PBS), and shaken for 3 h at room temperature. Supernatants were collected by centrifugation at 5000× g for 30 min and clarified by filtration with Whatman no. 2 paper and another centrifugation at 12,000× g for 3 min at 10 °C. Then, the content of surface protein in the collected supernatants was quantified by the Bradford assay [22 (link)]. Then, the proteins were separated by electrophoresis and trypsin hydrolysis. After desalination and lyophilization, samples were ready for analysis. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification and Gene Ontology (GO) analysis, eukaryotic orthologous groups (KOG) functional classification, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted by the Beijing Genomics Institute (BGI, Shenzhen, China). In brief, proteins were separated through LC (eksigent ultra 2D, SCIEX, Framingham, MA, USA) and detected by Triple TOF 5600 (SCIEX, Framingham, MA, USA). The MS/MS data were aligned with A. flavus 3357 proteome (NCBI database).