TY - JOUR
T1 - Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis
AU - Han, Wenjuan
AU - Yamauchi, Mika
AU - Hasegawa, Urara
AU - Noda, Masanori
AU - Fukui, Kiichi
AU - van der Vlies, André J.
AU - Uchiyama, Susumu
AU - Uyama, Hiroshi
N1 - Publisher Copyright:
© 2014 The Society for Biotechnology, Japan.
PY - 2015/5/1
Y1 - 2015/5/1
N2 - 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.
AB - 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.
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U2 - 10.1016/j.jbiosc.2014.10.018
DO - 10.1016/j.jbiosc.2014.10.018
M3 - Article
C2 - 25468419
AN - SCOPUS:84925163869
SN - 1389-1723
VL - 119
SP - 505
EP - 510
JO - Journal of Bioscience and Bioengineering
JF - Journal of Bioscience and Bioengineering
IS - 5
ER -