Protein digestion is one of the most important processes in proteomic analysis. Here, we report microwave-assisted protein digestion in a plate well, which allows for facile sampling as well as rapid protein digestion based on the combination of highly stable enzyme immobilization and 3D printing technologies. Trypsin (TR) was immobilized on polystyrene-based nanofibers via an enzyme coating (EC) approach. The EC with stabilized TR activity was assembled with the 3D-printed structure in the plate well (EC/3D), which provides two separated compartments for the solution sampling and the TR-catalyzed protein digestion, respectively. EC/3D can effectively prevent the interference of sampling by accommodating EC in the separated compartment from the sampling hole in the middle. EC/3D in the plate well maintained its protein digestion performance under shaking over 160 days. Microwave irradiation enabled the digestion of bovine serum albumin within 10 min, generating the MALDI-TOF MS results of 75.0% sequence coverage and 61 identified peptides. EC/3D maintained its protein digestion performance under microwave irradiation after 30 times of recycled uses. EC/3D in the plate well has demonstrated its potential as a robust and facile tool for the development of an automated protein digestion platform. The combination of stable immobilized enzymes and 3D-printed structures can be potentially utilized not only for the protein digestion, but also for many other enzyme applications, including bioconversion and biosensors. (Figure Presented).
Bibliographical noteFunding Information:
This research was supported by Global Research Laboratory Program (2014K1A1A2043032) and Nano·Material Technology Development Program (2014M3A7B4052193) through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT. This work was also supported by Business for Cooperative R&D between Industry, Academy, and Research Institute funded by Korea Small and Medium Business Administration in 2015 (C0298433). This research was also supported by the Korea Institute of Energy Technology Evaluation and Planning (KETEP) and the Ministry of Trade, Industry & Energy (MOTIE) of the Republic of Korea (No. 20142020200980). The authors would like to thank Eunjung Son for her help in the revision process.
© 2017 American Chemical Society.
ASJC Scopus subject areas
- Analytical Chemistry