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

Authors :: Han W
Yamauchi M
Hasegawa U
Noda M
Fukui K
van der Vlies AJ
Uchiyama S
Uyama H
Source :: Journal of bioscience and bioengineering [J Biosci Bioeng] 2015 May; Vol. 119 (5), pp. 505-10. Date of Electronic Publication: 2014 Nov 20.
Publication Year :: 2015
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.<br /> (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Subjects :: Chromatography, Liquid
Enzyme Stability
Hydrogen-Ion Concentration
Mass Spectrometry
Peptide Fragments chemistry
Peptide Fragments metabolism
Polystyrenes chemistry
Tandem Mass Spectrometry
Temperature
Aldehydes chemistry
Immobilized Proteins chemistry
Pepsin A chemistry
Pepsin A metabolism
Peptide Fragments analysis
Polymethacrylic Acids chemistry

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