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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.
Journal of Separation Science 06/2011; · 2.73 Impact Factor
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ABSTRACT: The selective enrichment of phosphopeptides with good reproducibility is vital for the in-depth study of the phosphoproteome. Herein, we presented a novel method to prepare monolithic Ti(4+) or Zr(4+) immobilized metal affinity chromatography (IMAC) columns. Since succinimide was of high reaction activity with nitrilotriacetic acid (NTA) than traditional epoxy group, through the reaction of succinimide group on the monolithic matrix with nitrilotriacetic acid, the preparation of monolithic IMAC columns became facile. By the developed new method, not only the time required for Ti(4+) or Zr(4+) immobilization could be shortened from 10 to 1 h, but also the RSD of obtained phosphopeptide peak area was below 13%, even with IMAC columns prepared in different batches (n=3). With such monolithic Ti(4+) - and Zr(4+) -IMAC columns, ten and seven phosphopeptides were effectively identified from the digests of a mixture of β-casein and BSA, even with the molar ratio as low as 1:100, respectively. The phosphopeptide recovery was over 73%, and the loading capacity was over 0.8 μmol/mL. Compared with commercial IMAC beads, the monolithic IMAC columns provide outstanding reproducibility, good selectivity, large loading capacity, and high recovery, which demonstrates that such monolithic IMAC columns might facilitate the in-depth analysis of phosphoproteomes.
Journal of Separation Science 05/2011; · 2.73 Impact Factor
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ABSTRACT: A novel detection method for the analysis of multi-phosphopeptides using microcolumn high performance liquid chromatography-electrospray ionization tandem mass spectrometry (microHPLC-ESI-MS/MS) was proposed by the dephosphorylation treatment of alkaline phosphatase (AP). After the selective enrichment by a microcolumn packed with TiO2, phosphopeptides from the tryptic digests of beta-casein were dephosphorylated by AP. Through the removal of phosphate groups, the detection of multi-phosphopeptides according to the non-phosphorylated ones was achieved by ESI-MS/MS. By comparing the chromatograms before and after the AP treatment, mono-phosphopeptides were identified based on the relative molecular mass (Mr) difference of 80. Furthermore, since more peaks appeared after the treatment, the existence of multi-phosphopeptides was proven. By controlling the treatment procedure, the partial dephosphorylation of multi-phosphopeptides was performed, and the multi-phosphorylated stees of the digest of beta-casein were found to be on serine residues at the possible sites of 17, 18 and 19.
Se pu = Chinese journal of chromatography / Zhongguo hua xue hui 08/2007; 25(4):453-6.
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ABSTRACT: A novel monolithic enzymatic microreactor was prepared in the fused-silica capillary by in situ polymerization of acrylamide (AA), N-acryloxysuccinimide (NAS) and ethylene dimethacrylate (EDMA) in the presence of a binary porogenic mixture of dodecanol and cyclohexanol, which could offer very low back pressure, enabling the fast digestion of proteins. The performance of the monolithic microreactor was demonstrated by digesting cytochrome c at high flow rate, and the comparisons between the in-solution digestion and on-column reaction were made by a nano-high performance liquid chromatography-mass spectrometry (nano-HPLC-MS) system. The performance of the monolithic microreactor was demonstrated with the digestion of cytochrome c at the fast flow rate of 1 microL/min, which afforded a residence time of 7s, yielding a sequence coverage of 54.81% using strict multiple database searching thresholds. Future more, a mixture of four standard proteins was digested and analyzed using the on-line digestion and nano-HPLC-MS system. The results showed the promising of such a system in the analysis of protein mixture.
Journal of Chromatography 03/2006; 1106(1-2):165-74. · 4.53 Impact Factor
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ABSTRACT: Extracts of Adinandra nitida leaves, known as Shiyacha in China, were analyzed by monolithic columns of capillary electrochromatography (CEC). To obtain good resolution within a short time, stepwise gradient elution of CEC was employed, and the effects of experimental parameters, such as the buffer, the gradient conditions, and the mode of injection were studied systematically. Under optimized conditions, analysis could be accomplished in 25 min on a monolithic rod of macroporous poly(butyl methacrylate-co-ethylene dimethacrylate). With two identified flavonoids, epicatechin and apigenin, as markers, a quality control method for Shiyacha and its relevant products was established. The calibration curves exhibited good linear behavior over the concentration range of two orders of magnitude. On combination with an on-line concentration technique, the detection limit of flavonoids could be decreased to 25 ng for apigenin.
Journal of Separation Science 06/2005; 28(8):774-9. · 2.73 Impact Factor
