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.

Initially, a poly (glycidyl methacrylate-co-acrylamide-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 chromatography-electrospray ionization-mass spectrometry successfully, showing its practicability for the high throughput analysis of complex samples.

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling.

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29 µg, was analyzed, and sequence coverages for BSA, myoglobin, ß-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.

The Effect of Monolith Properties on the Digestion Performance of Monolith-Based Immobilized Enzyme Microreactor.

In this work, two kinds of monolith-based immobilized enzyme microreactors (IMER) were compared in terms of monolith properties and digestion performance. Two monoliths were prepared involving poly (tetraethoxysiLane-co-3-aminopropyltriethoxysilane) (poly (TEOS-co-APTES)) monolith and poly (N-acryloxysuccinimide-co-acrylamide-co-ethylene dimethacrylate) (poly (NAS-co-AA-co-EDMA)) monolith for covalent trypsin immobilization. A higher chromatographic permeability constant 66.7 was obtained from the poly (TEOS-co-APTES) monolith in comparison with only 4.37 for poly (NAS-co-AA-co-EDMA) monolith, and the porous property of monolith measured by N2 adsorption/desorption and mercury intrusion porosimetry also indicated that poly (TEOS-co-APTES) monolith has more pores than poly (NAS-co-AA-co-EDMA) monolith. On the other hand, the digestion results demonstrated that the more efficient IMER was obtained by immobilizing trypsin on the poly (TEOS-co-APTES) monolith even if a less amount of immobilized trypsin determined by the Bradford method than poly (NAS-co-AA-co-EDMA) monolith. Therefore, the capacity of trypsin immobilization of monolith was not the exclusive factor to determine the digestion performance of IMER. Physical properties-penetrability and pore distribution of monolith showed a crucial effect on the digestion performance of IMER.

Facile synthesis of hybrid hollow mesoporous nanospheres with high content of interpenetrating polymers for size-selective peptides/proteins enrichment.

A facile synthesis of polymer-inorganic hybrid hollow mesoporous nanospheres was developed based on the entrapment of a dissolved polymer core template in the framework during the assembly process of the hybrid hollow nanospheres for efficient and size-selective enrichment of target peptides/proteins from complex biosamples.