Journal
JOURNAL OF BIOSCIENCE AND BIOENGINEERING
Volume 119, Issue 5, Pages 505-510Publisher
SOC BIOSCIENCE BIOENGINEERING JAPAN
DOI: 10.1016/j.jbiosc.2014.10.018
Keywords
Monolith; Pepsin; Online digestion; Thermally induced phase separation; Polymethacrylate
Funding
- Japan Society for the Promotion of Science [25121722, 25288090]
- Ministry of Education, Culture, Sports, Science and Technology (MEXT)
- Manufacturing Technology Research of Biologics from METI
- China Scholarship Council (CSC)
- Grants-in-Aid for Scientific Research [25288090] Funding Source: KAKEN
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Polymer-based monoliths with interconnected porous structure have attracted much attention as a high-performance stationary phase for online digestion liquid chromatography-mass spectrometry (LC-MS) system. In this study, a poly(glycidyl methacrylate-co-methyl methacrylate) (PGM) monolith prepared via thermally induced phase separation (TIPS) was used as a solid support to covalently immobilize pepsin. The PGM monolith was modified with aminoacetal to yield an aldehyde-bearing (PGM-CHO) monolith. Pepsin was immobilized onto the PGM-CHO monolith via reductive amination. The immobilized pepsin showed better pH and thermal stability compared with free pepsin. Furthermore, the PGM-CHO monolith modified with pepsin was applied for online protein digestion followed by LC-MS and LC-MS/MS analyses. As a result, a larger number of peptides are reproducibly identified compared to those by polystyrene/divinylbenzene particle (POROS)-based online pepsin column. (C) 2014, The Society for Biotechnology, Japan. All rights reserved.
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