Abstract
The goal of this paper was to develop an in-line immobilized enzyme reactor (IMER) integrated into a capillary electrophoresis platform. In our research, we created the IMER by adsorbing trypsin onto the inner surface of a capillary in a short section. Enzyme immobilization was possible due to the electrostatic attraction between the oppositely charged fused silica capillary surface and trypsin. The reactor was formed by simply injecting and removing trypsin solution from the capillary inlet (~1–2 cms). We investigated the factors affecting the efficiency of the reactor. The main advantages of the proposed method are the fast, cheap, and easy formation of an IMER with in-line protein digestion capability. Human tear samples were used to test the efficiency of the digestion in the microreactor.
Highlights
Capillary Electrophoresis System.The bottom-up approach used in proteomics is one of the most-often applied methods for the analysis of proteins
The sample is first mixed with a proteolytic enzyme to cut the long chains of proteins into smaller parts; the obtained peptides are separated by high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) before their detection with tandem mass spectrometry (MS/MS) [1]
This implies that a compromise has to be made, since the acidic background electrolytes (BGE) commonly utilized for bottom-up proteomic studies in a CE-MS setting inhibit tryptic activity
Summary
The bottom-up approach used in proteomics is one of the most-often applied methods for the analysis of proteins. In this workflow, the sample is first mixed with a proteolytic enzyme (e.g., trypsin) to cut the long chains of proteins into smaller parts; the obtained peptides are separated by high-performance liquid chromatography (HPLC) or capillary electrophoresis (CE) before their detection with tandem mass spectrometry (MS/MS) [1]. In the standard protein digestion procedure, only a low concentration of the proteolytic enzyme is allowed in order to minimize the autolysis of the enzyme; the enzymatic reaction requires long incubation times (typically overnight) to complete the digestion [1,2]. The necessity of such long incubation times is the bottleneck for fast, high-throughput, bottom-up proteomic analyses
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