Journal of Chromatography A, 1209 (2008) 95–103 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Evaluation of various immobilized enzymatic microreactors coupled on-line with liquid chromatography and mass spectrometry detection for quantitative analysis of cytochrome c A. Cingöz, F. Hugon-Chapuis ∗ , V. Pichon Department of Environmental and Analytical Chemistry (UMR CNRS 7121), Ecole Supérieure de Physique et de Chimie Industrielles, 10 rue Vauquelin, 75231 Paris Cedex 05, France article info Article history: Received 29 April 2008 Received in revised form 21 August 2008 Accepted 21 August 2008 Available online 11 September 2008 Keywords: IMER Trypsin On-line digestion Cytochrome c LC–MS/MS 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 esti- mating 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 sup- port (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. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Real improvements were achieved in proteomic field during the last decades including the discovery of biomarkers that are nowadays a very active area of this field [1]. The identification of proteins is commonly achieved by peptides mapping using mass spectrometry (MS) [2]. However, this identification method requires a proteolytic digestion before the chromatographic separa- tion of peptides and their identification by mass spectrometry [2,3]. Trypsin is the most widely used enzyme for fragmenting proteins. This enzyme cleaves selectively proteins on the C-terminal side of the basic amino acid residues lysine and arginine, except when they are immediately followed by proline. Digestions are com- monly performed in solution during several hours [4,5]. However, the enzyme/protein ratio cannot be increased in order to reduce the digestion time because an autoprotolysis of the proteolytic enzyme can occur. The resulting fragments of this autoprotolysis can then interfere during the detection [6,7]. To overcome this drawback, the proteolytic enzyme can be immobilized on a functionalized solid support to prevent the autoprotolysis, thus giving rise to an effi- cient digestion in a reduced time [8]. In addition, as the stability of the enzyme is enhanced, immobilized enzyme reactors (IMERs) ∗ Corresponding author. Tel.: +33 1 4079 4673; fax: +33 1 4079 4776. E-mail address: florence.hugon@espci.fr (F. Hugon-Chapuis). can be easily reused, thus decreasing the cost of this digestion step. The nature of the functionalized immobilization support is a key parameter controlling the performances of the IMER. Indeed, it should be biologically inert, easily activated, mechanically stable, uniform in particle size with large pores to favour a good accessi- bility of enzyme. Moreover, this support should be hydrophilic to avoid non-specific hydrophobic interactions and make easier the transfer of peptides from IMER to the chromatographic system. Several supports allowing a chemical or a physical bonding of enzymes were already used for enzymes immobilization [9]. Physi- cal immobilization techniques as encapsulation of enzymes within a gel have been reported [10]. Adsorption of enzymes on a sup- port was also used with the advantage to keep the structure and the activity of enzyme. However a leaching of enzymes may occur [11,12]. The most widely used immobilization technique involves a covalent bonding of enzymes on a functionalized solid support preventing the leakage and improving the IMER lifetime. IMERs can be easily integrated to the analytical system allowing an automated procedure as recently reviewed [13]. Trypsin-based IMERs were specially developed for proteomic studies, to be inte- grated to the LC–MS analysis [14]. The most used IMER is a commercial device (Porozyme) developed by PerSeptive in the last decade and composed of trypsin immobilized on a cross-linked poly(stryrene-divinylbenzene) packed in a precolumn to be cou- pled in-line with the LC–MS system for the analysis of various 0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2008.08.120