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Updated: Jun 30, 2026
Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor
Published on: April 6, 2016
Evaluation of various immobilized enzymatic microreactors coupled on-line with liquid chromatography and mass
A Cingöz1, F Hugon-Chapuis, V Pichon
1Department of Environmental and Analytical Chemistry (UMR CNRS 7121), Ecole Supérieure de Physique et de Chimie Industrielles, 10 rue Vauquelin, 75231 Paris Cedex 05, France.
Abstract:
An in-line procedure for protein analysis using a trypsin-based immobilized enzymatic reactor (IMER) coupled to LC-MS/MS has been developed. Various IMERs were synthesized and characterized by estimating the digestion yield of a pattern peptide in UV detection. Laboratory-made IMERs were optimized by studying the effect of different parameters as the nature of the functionalized immobilization support (silica, agarose), the amount of immobilized trypsin and the binding density. The potential of the laboratory-made IMERs were compared with a batch digestion and with a commercial trypsin-based IMER. The laboratory-made IMER based on CNBr-activated Sepharose showed the best performances in terms of digestion yields, digestion time, price and repeatability (RSD<4%). Cytochrome c was then digested on this IMER and used in-line with LC-MS. The target protein was easily recognized by the Mascot database until 17pmol injected.
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