Less common applications of monoliths: I. Microscale protein mapping with proteolytic enzymes immobilized on monolithic supports

Abstract

This review summarizes the recent contributions to the rapidly growing area of immobilized enzymes employing both silica and synthetic polymer-based monoliths as supports. Focus is mainly on immobilized proteolytic enzyme reactors designed for studies in proteomics. Porous monoliths emerged first as a new class of stationary phases for HPLC in the early 1990s. Soon thereafter, they were also used as supports for immobilization of proteins and preparation of both stationary phases for bioaffinity chromatography and enzymatic reactors. Organic polymer-based monoliths are typically prepared using a simple molding process carried out within the confines of a "mold" such as chromatographic column or capillary. Polymerization of a mixture comprising monomers, initiator, and porogenic solvent affords macroporous materials. In contrast, silica-based monoliths are first formed as a rigid rod from tetraalkoxysilane in the presence of PEG and subsequently encased with a plastic tube. Both types of monolith feature large through-pores that enable a rapid flow-through. Since all the solutions must flow through the monolith, the convection considerably accelerates mass transfer within the monolith. As a result, reactors including enzyme immobilized on monolithic support exhibit much higher activity compared to the reactions in solution.

Synthesis of boronate-silica hybrid affinity monolith via a one-pot process for specific capture of glycoproteins at neutral conditions

“…With the unique properties of simplicity of preparation, rapid mass-transfer rate, low backpressure, and versatile surface modification, monolithic materials have been more and more attractive and widely used in proteomic analysis [21,22]. Since Malik et al [23] first prepared a C 18 -incorprated hybrid monolith, the organic-silica hybrid monolith has drawn more attentions in the separation field [24,25], which combines the advantages of both organic monolith and inorganic monolith, such as easily fabricated, less shrinkage and good pH stability.…”

Development of Core-shell Magnetic Mesoporous SiO2 Microspheres for the Immobilization of Trypsin for Fast Protein Digestion

“…Meanwhile, the sample volume is only 40 µ L per analysis. Moreover, no trypsin autolysis peaks were observed from mass spectra in the microwave-assisted digestion method, which demonstrated that enzyme immobilization technique can overcome the trypsin autolysis (Dogruel et al, 1995;Nelson, 1997;Gobom et al, 1997; Jiang et al, 2000; Ekstrom et al, 2000;Peterson et al, 2002;Licklider et al, 1995;Ma et al, 2007;Svec, 2006). Since the magnetic microspheres are excellent microwave absorbers; and thus greatly improve the efficiency of protein digestion.…”

“…In both approaches, to obtain detailed structural information, proteins are selectively cleaved into smaller polypeptide fragments by controlled chemical or enzymatic reactions Wolters et al, 2001;Zhu et al, 2003 (Nalivaeva and Turner, 2001). The immobilized enzyme has been adopted to characterize the proteins with benefits from the reusability and stability of enzyme, the higher digestion efficiency of protein analytes, and no enzyme autolysis products (Dogruel et al, 1995;Nelson, 1997;Gobom et al, 1997; Jiang et al, 2000; Ekstrom et al, 2000;Peterson et al, 2002;Licklider et al, 1995;Ma et al, 2007;Svec, 2006). The main approach of enzyme immobilization is covalent binding.…”

Less common applications of monoliths

“…Their applications in a variety of liquid chromatographic modes including high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC) has recently been described in several reviews [12][13][14][15][16][17][18][19][20][21][22][23][24][25] and books [11,26]. However, the less common applications of monolithic materials that include supports for solid phase and combinatorial synthesis [27][28][29], scavengers [30,31], carriers for immobilization of enzymes [32][33][34], static mixers [35], thermally responsive gates and valves [36][37][38], as well as solid phase extractors and pre-concentrators [39] are escaping the awareness of the scientific community. The recently started series of review articles aims at popularization of these applications.…”

“…The recently started series of review articles aims at popularization of these applications. So far, this series detailed achievements in microscale protein mapping with proteolytic enzymes immobilized on monolithic supports and in preconcentration and solid-phase extraction [34,39]. Present contribution focuses on use of monolithic materials in gas chromatography.…”