Preparing a metal-ion chelated immobilized enzyme reactor based on the polyacrylamide monolith grafted with polyethylenimine for a facile regeneration and high throughput tryptic digestion in proteomics

Abstract

Initially, a poly (glycidyl methacrylate-coacrylamide-co-methylenebisacrylamide) monolith was prepared in the 100 μm i.d. capillary, and then was grafted with polyethylenimine (Mw, ∼25,000) for adsorbing Cu 2+ , followed by chelating trypsin. As a result, efficient digestion for BSA (100 ng/μL) was completed within 50 s via such immobilized enzyme reactor (IMER); yielding 47% sequence coverage by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. Compared with the conventional method for preparing the metal-ion chelated IMER, the regeneration of such IMER can be achieved facilely by the respective 30 min desorption and re-adsorption of trypsin, and 51% sequence coverage was obtained for 50 s BSA digestion after regeneration. BSA down to femtomole was also efficiently digested by the prepared regenerable IMER. Meanwhile, after the consecutive digestion of myoglobin and BSA, there was not any mutual interference for both during MALDI-TOF MS identification, indicating the low nonspecific adsorption of such regenerable IMER. To test the applicability of regenerable IMER for complex sample profiling, proteins (150 ng) extracted from Escherichia coli were digested within 80 s by the regenerable IMER and further analyzed by nanoreversed phase liquid chromatographyelectrospray ionization-mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.

Flow‐through immobilized enzyme reactors based on monoliths: I. Preparation of heterogeneous biocatalysts

“…Scheme of preparation of regenerable IMER . Reprinted with permission of W u et al . Copyright 2011 Springer‐Verlag.…”

“…However, this disadvantage can also be considered as a possibility to prepare the regenerable heterogeneous systems. Some interesting examples of preparation of monolithic biocatalysts were found in current literature .…”

“…Recently, Zhang's group suggested the preparation of trypsin heterogeneous biocatalyst based on macroporous organic–inorganic hybrid silica , as well as on poly(glycidyl methacrylate‐co‐acrylamide‐co‐methylene‐bis‐acrylamide) monolithic capillary columns using metal‐ion chelation adsorption. In first case, the hybrid monolithic support containing iminodiacetic acid moiety was prepared and treated with 50 mM solution of CuSO 4 for 1 h. The activated monolithic column was filled with 2 mg/mL trypsin solution by pumping it through a matrix and stored for 2 h. Finally, nonadsorbed enzyme was removed by washing with 100 mM sodium phosphate buffer, pH 8.0.…”

“…In the case of preparation of heterogeneous biocatalysts based on organic polymer monolith, the prepared kind of polyacrylamide monolith was grafted with PEI (M W 25000) for adsorbing Cu 2+ ions (Fig. ) . The chelation was done by flushing of monolith with solution of Cu 2+ for 1 h at 25°C, pH ∼ 4, followed by washing with 2 M NaCl (pH ∼ 3) for 10 min.…”

Sequential optimization of methotrexate encapsulation in micellar nano-networks of polyethyleneimine ionomer containing redox-sensitive cross-links

“…24 The polymer has a high content of amine nitrogen able to donate electrons and coordinate with transition metal ions. 25,26 This property may be applied for the immobilization of proteins and enzymes, [27][28][29] anchoring metal nanoparticles, 30 or drug delivery. 31,32 Different authors have reported ionomer complex micelles of PEI grafted with methoxypolyethylene glycol (mPEG) and anionic surfactants, 9,33 metal ions (AuCl 3 or PtCl 6 2-), 26,34 oligonucleotides, 35 or plasmid DNA.…”