While significant progress has been made in understanding the digestive process of Penaeus vannamei, the specific sequence of enzyme participation in the protein digestion process is still unknown. Since the whiteleg shrimp is a major aquaculture product in Mexico, this knowledge becomes important to improve the culture process. To accomplish this, the digestive peptidases abundance and activity in the shrimp digestive gland were analyzed at three different times, each representing an essential stage in the digestion process. Zymograms and Tandem Mass Tags (TMT) were used for activity and relative quantitative proteomic analysis.
Midgut glands were sampled three times during the digestion process: preprandial, postprandial 1 h, and postprandial 3 h. Samples were examined by activity tests (zymograms) and proteomics. Proteins were digested with MS-grade trypsin, peptides labeled with Amine-reactive TMT-10plex and fractionated by SCX. Protein identification and relative quantitation analysis was performed by SPS-MS3 using an Orbitrap and raw-data were processed with Proteome Discoverer 2.1 using Amanda, Sequest-HT, and Mascot search engines against the P. vannamei proteome database (UP000283509).
Results showed that serine peptidases (trypsin and chymotrypsin isoforms) were active overall during the digestion process, but differences in relative abundances were detected in only two trypsins, trypsin 2 which decreased at postprandial 1 h and 3 h, and trypsin 1 which increased at postprandial 1 h and decreased again in postprandial 3 h, while both chymotrypsins increased their abundance at postprandial 3 h. Digestive cathepsin D (A0A423SAG9) and cathepsin L both showed significant changes in activity and abundance at preprandial (cathepsin D) and postprandial 3 h (cathepsin L). Two digestive metallopeptidases showed a similar change with their maximal abundances during postprandial 3 h, and no activity during postprandial (3 h).
Even though trypsin and chymotrypsins are the most important peptidases in the digestive process, regulated peptidases were cathepsins, cathepsin D being most abundant at preprandial, whereas cathepsin L during postprandial. Metallopeptidases had similar abundance in the three times but showed no activity at postprandial.