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Chararacterization of a monolithic immobilized trypsin microreactor with on‐line coupling to ESI‐MS
Author(s) -
Křvenková Jana,
Bilková Zuzana,
Foret František
Publication year - 2005
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.200500171
Subject(s) - chemistry , chromatography , bioreactor , trypsin , glycidyl methacrylate , monolithic hplc column , mass spectrometry , immobilized enzyme , monolith , covalent bond , microreactor , analytical chemistry (journal) , polymerization , high performance liquid chromatography , organic chemistry , enzyme , polymer , catalysis
The preparation and characterization of a miniaturized trypsin reactor using on‐line coupling with an ESI‐TOF mass spectrometer are described. L ‐1‐Tosylamido‐2‐phenylethyl chloromethyl ketone‐trypsin was covalently immobilized on poly(glycidyl methacrylate‐ co ‐ethylene dimethacrylate) monolith prepared in a 75 μm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one‐ or a multistep reaction. For on‐line protein digestion‐MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on‐line flow through the system with flow rates of 50–300 nL/min. The resulting protein consumption was in the atto‐ to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25°C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37°C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in‐solution protocols.