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J Biosci Bioeng. 2015 May;119(5):505-10. doi: 10.1016/j.jbiosc.2014.10.018. Epub 2014 Nov 20.

Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis.

Author information

1
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
2
U-medico, Inc., Suita 565-0871, Japan.
3
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan; Frontier Research Base for Young Researchers and Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
4
U-medico, Inc., Suita 565-0871, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
5
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
6
Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan. Electronic address: uyama@chem.eng.osaka-u.ac.jp.

Abstract

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.

KEYWORDS:

Monolith; Online digestion; Pepsin; Polymethacrylate; Thermally induced phase separation

PMID:
25468419
DOI:
10.1016/j.jbiosc.2014.10.018
[Indexed for MEDLINE]

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