Evaluation of various immobilized enzymatic microreactors coupled on-line with liquid chromatography and mass spectrometry detection for quantitative analysis of cytochrome c

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 17 pmol injected.