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In this work, a novel MoS2-based immobilized trypsin reactor was designed and prepared. Pyrene-l-butyric acid was first assembled onto MoS2 nanosheets via the strong pi-pi stacking and then trypsin was covalently immobilized onto the nanocomposite supports through amidation reaction. Compared with traditional in-solution digestion, higher sequence coverage (84%) and shorter time (5 min) could be achieved by the novel trypsin reactor during the digestion of BSA. The excellent performances of as-prepared trypsin reactor can be mainly attributed to the designed novel structure of the composites with high surface area resulting in high enzyme loading. In addition, strong reusability, good reproducibility and long storage of the trypsin reactor were also obtained. The novel immobilized trypsin reactor was further applied in large-scale proteomics research. The proteins extracted from HeLa cells and Amygdalus Pedunculata Pall. kernels were chosen to evaluate the digestion performance for the novel MoS2-based immobilized trypsin reactor, and the experimental results showed that the number of identified proteins from complex real bio-samples with 1 h immobilized tryptic digestion was slightly more than that obtained by 12 h in-solution digestion. The above results demonstrated that the protein digestion with our novel MoS2-based immobilized trypsin reactor is superior to the conventional protein digestion with free trypsin. Moreover, this simple, fast tryptic digestion method can effectively reduce the levels of artifacts in detection of oxidation and deamidation of peptides from proteins of Amygdalus Pedunculata Pall. kernels. Also, results of Gene Ontology analysis give an explanation for the good survival of Amygdalus Pedunculata Pall. in harsh desert environments from proteomics points of view. Therefore, the novel 2D-MoS2-based immobilized trypsin is potentially suitable for the high throughput proteome analysis and opening up a new avenue for Molybdenum disulfide in proteomics field.