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

Wenjuan Han; Mika Yamauchi; Urara Hasegawa; Masanori Noda; Kiichi Fukui; André J van der Vlies; Susumu Uchiyama; Hiroshi Uyama

doi:10.1016/j.jbiosc.2014.10.018

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

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

Authors

Wenjuan Han¹, Mika Yamauchi², Urara Hasegawa³, Masanori Noda⁴, Kiichi Fukui⁵, André J van der Vlies¹, Susumu Uchiyama⁵, Hiroshi Uyama⁶

Affiliations

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.

PMID: 25468419
DOI: 10.1016/j.jbiosc.2014.10.018

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

Aldehydes
Immobilized Proteins
Peptide Fragments
Polymethacrylic Acids
Polystyrenes
polymethacrylic acid
Pepsin A