Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

• Published in: Analytica chimica acta 2010-03, Vol.663 (2), p.198-205
• Main Authors: Dartiguenave, Catherine, Hamad, Hussein, Waldron, Karen C.
• Format: Article
• Language: English
• Subjects: Analytical chemistry; Bioreactors; Calcium - chemistry; Capillarity; Capillary electrophoresis; Chemistry; Chromatographic methods and physical methods associated with chromatography; Controlled pore glass; Digestion; Electrophoresis, Capillary; Enzymes; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Exact sciences and technology; Glass; Glass - chemistry; Hydrolysis; Immobilization; Immobilized enzyme reactor; Ions - chemistry; Isothiocyanates - chemistry; Kinetics; Mapping; Other chromatographic methods; Particle Size; Peptide Mapping; Peptides; Peptides - analysis; Porosity; Proteins - analysis; Surface Properties; Trypsin; Trypsin - chemistry; Trypsin - metabolism; β-Casein
• ISSN: 0003-2670; 1873-4324
• DOI: 10.1016/j.aca.2010.01.042

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca 2+ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC–CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage ( ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).