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 41 -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)-coethylene 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 41 -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 41 -IMAC monolith was used for phosphopeptide analysis and showed good selectivity in phosphopeptide enrichment with matrix-assisted laser desorption ionization mass spectrometry detection. IntroductionIn recent years, protein phosphorylation has gained much attention because of its significant roles in cellular signal transduction processes [1,2]. However, the amount of phosphorylated proteins in eukaryotic cells often exists in substoichiometry level, which makes the identification of phosphorylation sites challenging [3]. To date, mass spectrometry (MS) analysis has become an effective and commonly used method for characterization of phosphoproteins [4]. To reduce the interference of non-phosphopeptides, selective enrichment strategies, such as immobilized metal ion affinity chromatography (IMAC) [5][6][7], metal oxide affinity chromatography (MOAC) [8,9], and ion exchange chromatography [10][11][12][13], are necessary before MS analysis.Recently, chip-based system has been introduced as a promising platform for phosphopeptide analysis, due to the benefits of low sample amount, high throughput, and easy integration [14]. Several microfluidic devices have been developed by integrating phosphopeptide enrichment with other functions, such as protein alkylation and reduction [15], protein digestion [16], peptide separation and on-line interface for MS instrument [17][18][19][20], which have shown great potentials in the field of phosphoproteome research. However, in these microchip devices, phosphopeptide enrichment components are usually fabricated by packing functionalized particles, such as TiO 2 or IMAC beads, into m...

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