title: Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides.
abstract: To meet the demands of protein phosphorylation study, immobilized zirconium ion affinity chromatography (Zr(4+)-IMAC) monolith was prepared by combining UV-initiated polymerization of monolithic support and subsequent photografting in both capillary columns and microchannels. Hydrophilic poly(2-hydroxyethyl methacrylate (HEMA)-co-ethylene dimethacrylate (EDMA)) monolithic support was prepared under UV irradiation at the wavelength of 365 nm with monomer HEMA, crosslinker EDMA and 2,2-dimethoxy-2-phenylacetophenone as photoinitiator in 1-decanol solution, which provides good biocompatibility and permeability for biomolecule analysis. To introduce chelating ligands, such as phosphate groups, on the pore surface of monolith for metal ion immobilization, photografting of ethylene glycol methacrylate phosphate with benzophenone as the photoinitiator was performed at 254 nm for 300 s. The grafting process and metal ion immobilization can be monitored by measuring the electroosmotic flow produced by the modified monolith, providing a quantitative evaluation of post-modification. This new method for the preparation of Zr(4+)-IMAC monolith simplifies the optimization of monolith preparation and avoids the time-consuming chemical modification process. Additionally, advantages include facile preparation in microdevices, easy regenerability and good reproducibility. After optimization, the microchip-based Zr(4+)-IMAC monolith was used for phosphopeptide analysis and showed good selectivity in phosphopeptide enrichment with matrix-assisted laser desorption ionization mass spectrometry detection.
authors.affiliation: Key Laboratory of Separation Science for Analytical Chemistry, National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, P. R. China; Graduate School of Chinese Academy of Sciences, Beijing, P. R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Wang
authors.forename: Jicheng
authors.initials: J
authors.lastname: Duan
authors.forename: Hongjiu
authors.initials: H
authors.lastname: Yu
authors.forename: Hongjiu
authors.initials: H
authors.lastname: Xu
authors.forename: Liang
authors.initials: L
authors.lastname: Zhao
authors.forename: Yu
authors.initials: Y
authors.lastname: Liang
authors.forename: Yichu
authors.initials: Y
authors.lastname: Shan
authors.forename: Lihua
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhen
authors.initials: Z
authors.lastname: Liang
authors.forename: Yukui
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008689:Methacrylates / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry*; D015040:Zirconium / Q000737:chemistry*
chemical_list: D008689:Methacrylates; D010710:Phosphates; D010748:Phosphopeptides; D011108:Polymers; C005044:hydroxyethyl methacrylate; C004919:ethylene dimethacrylate; D015040:Zirconium; C508958:ethylene glycol methacrylate phosphate