Monoliths with immobilized zirconium ions for selective enrichment of phosphopeptides

• Published in: Journal of separation science 2011-08, Vol.34 (16), p.2113-2121
• Main Authors: Wang, Hui, Duan, Jicheng, Xu, Hongjiu, Zhao, Liang, Liang, Yu, Shan, Yichu, Zhang, Lihua, Liang, Zhen, Zhang, Yukui
• Format: Article
• Language: English
• Subjects: affinity chromatography; Biomolecules; Chromatography, Affinity - instrumentation; Chromatography, Affinity - methods; Desorption; Enrichment; ethylene glycol; Hydrophobic and Hydrophilic Interactions; Immobilization; Immobilized zirconium ion affinity chromatography; ionization; Ions; Mass spectrometry; Methacrylates - chemistry; Monolith; new methods; Optimization; Phosphates; Phosphates - chemistry; Phosphopeptide; Phosphopeptides - chemistry; Phosphopeptides - isolation & purification; Photografting; Photoinitiators; Photopolymerization; Polymerization; Polymers - chemical synthesis; Polymers - chemistry; protein phosphorylation; Selective enrichment; ultraviolet radiation; Zirconium; Zirconium - chemistry
• ISSN: 1615-9306; 1615-9314; 1615-9314
• DOI: 10.1002/jssc.201100168

To meet the demands of protein phosphorylation study, immobilized zirconium ion affinity chromatography (Zr4+‐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 Zr4+‐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 Zr4+‐IMAC monolith was used for phosphopeptide analysis and showed good selectivity in phosphopeptide enrichment with matrix‐assisted laser desorption ionization mass spectrometry detection.