title: Pepsin immobilization on an aldehyde-modified polymethacrylate monolith and its application for protein analysis.
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.
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors.forename: Wenjuan
authors.initials: W
authors.lastname: Han
authors.affiliation: U-medico, Inc., Suita 565-0871, Japan.
authors.forename: Mika
authors.initials: M
authors.lastname: Yamauchi
authors.affiliation: 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.
authors.forename: Urara
authors.initials: U
authors.lastname: Hasegawa
authors.affiliation: U-medico, Inc., Suita 565-0871, Japan; Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Noda
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors.forename: Kiichi
authors.initials: K
authors.lastname: Fukui
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors.forename: André J
authors.initials: AJ
authors.lastname: van der Vlies
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan.
authors.forename: Susumu
authors.initials: S
authors.lastname: Uchiyama
authors.affiliation: Department of Applied Chemistry, Graduate School of Engineering, Osaka University, Suita 565-0871, Japan. Electronic address: uyama@chem.eng.osaka-u.ac.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Uyama
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D002853:Chromatography, Liquid; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D055767:Immobilized Proteins / Q000737:chemistry*; D013058:Mass Spectrometry; D010434:Pepsin A / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011109:Polymethacrylic Acids / Q000737:chemistry*; D011137:Polystyrenes / Q000737:chemistry; D053719:Tandem Mass Spectrometry; D013696:Temperature
chemical_list: D000447:Aldehydes; D055767:Immobilized Proteins; D010446:Peptide Fragments; D011109:Polymethacrylic Acids; D011137:Polystyrenes; C030613:polymethacrylic acid; D010434:Pepsin A
keywords: Monolith; Online digestion; Pepsin; Polymethacrylate; Thermally induced phase separation