Controlled enzyme-immobilisation on capillaries for microreactors for peptide mapping

Abstract

In the present paper, the covalent immobilisation of the digesting enzyme trypsin has been achieved through photo-immobilisation on a portion of a silica capillary, thus leading to the construction of a capillary electrophoretic (CE)-microreactor for peptide mapping. The CE-microreactor is characterised by being a single piece, thus ensuring no fluidic or electrical leakage. The enzyme was immobilised with a surface density of 15.8 microg/cm(2), the stability was high (80% after 38 days) and the rate of conversion was 0.2 ng/s. On-line protein mapping was tested with proteins of different dimensions, showing competitiveness in terms of time (completed map within 15 min) and exhaustive maps of small proteins. The results of the CE-microreactor and the potential to immobilise biocomponents easily on a desired portion of the capillary indicate further developments towards the construction of a variety of miniaturised enzymatic screening devices for high-throughput screening analysis.

Recent developments in capillary electrophoresis and capillary electrochromatography of peptides

“…Covalent binding of the digesting enzyme trypsin has been achieved through photoimmobilization of this enzyme to a portion of an FS capillary with the surface density of 15.8 mg/cm 2 [68]. The immobilized enzyme exhibited high stability (80% after 38 days) and conversion rate of 0.2 ng/s.…”

“…CZE and HPLC peptide mapping have been used for characterization of insoluble avian eggshell matrix proteins; better CZE separation and higher number of resolved peaks were obtained in FS capillary dynamically coated with the triblock copolymer Pluronic F127 [77] than in the bare FS capillary [283]. In some advanced systems, CE and CEC peptide mapping is online connected with previous protein digestion by enzymes (trypsin, pepsin) immobilized in microreactors directly coupled to separation capillary [68][69][70].…”

Immobilized trypsin systems coupled on-line to separation methods: Recent developments and analytical applications

“…Bossi et al [64] proposed the covalent immobilization of trypsin on a portion of silica capillary through a photo-coupling reaction, leading to the construction of a CE-microreactor for peptide mapping. The bioreactor was characterized by being a single piece, thus ensuring no fluidic or electrical leakage typical of the reactors constructed as multiassemblies.…”

A hydrophilic immobilized trypsin reactor with N-vinyl-2-pyrrolidinone modified polymer microparticles as matrix for highly efficient protein digestion with low peptide residue

“…Furthermore, it was difficult to be integrated online with HPLC-MS to achieve high throughput proteome analysis [2,3]. Therefore, to solve these problems, recently immobilized enzyme reactors (IMERs) have been paid much attention to, by attaching enzymes on various supports, such as monoliths [4][5][6], microbeads or particles [7,8], magnetic nanoparticles [9][10][11], carbon nanotubes [12], fusedsilica capillaries [13], and membranes [14]. Although IMERs could facilitate highly efficient protein digestion and on-line coupling with HPLC-MS, peptide residue or carry over on matrices caused by the nonspecific adsorption brings great challenges for the application of IMERs, especially for the quantitative proteome analysis.…”