Hydrophilic monolith based immobilized enzyme reactors in capillary and on microchip for high-throughput proteomic analysis

A novel kind of hydrophilic monolith based immobilized enzyme reactors (IMERs) was prepared both in UV-transparent capillaries and on glass microchips by the photopolymerization of N-acryloxysuccinimide and poly(ethylene glycol)diacrylate, followed by trypsin immobilization. The performance of capillary IMERs for protein digestion was evaluated by the digestion of myoglobin with the residential time from 12s to 71 s. With μRPLC-ESI-MS/MS analysis, the obtained sequence coverages were all over 80%, comparable to that obtained by in-solution digestion for 12 h. The nonspecific absorption of BSA on monolithic support was evaluated, and no obvious protein residue was observed by a fluorescence assay. Moreover, no carry-over of the digests on the capillary IMER was found after the digestion of myoglobin (24 μg) and BSA (9 μg), which further demonstrated the good hydrophilicity of such matrix. In addition, an integrated microchip-based system involving on-line protein digestion by microchip-based IMER, peptides separation by nanoRPLC and identification by ESI-MS/MS was established, by which a mixture of standard proteins and one RPLC fraction of Escherichia coli extract were successfully identified, indicating that the hydrophilic monolith based IMER might provide a promising tool for high-throughput proteomic analysis.     

Trypsin immobilization on an ethylenediamine-based monolithic minidisk for rapid on-line peptide mass fingerprinting studies

The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The micro-immobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10 min only.     

Development of an In-Line Enzyme Reactor Integrated into a Capillary Electrophoresis System

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.     

Immobilized trypsin systems coupled on-line to separation methods: recent developments and analytical applications

The ability to rapidly and efficiently digest and identify an unknown protein is of great utility for proteome studies. Identification of proteins via peptide mapping is generally accomplished through proteolytic digestion with enzymes such as trypsin. Limitations of this approach consist in manual sample manipulation steps and extended reaction times for proteolytic digestion. The use of immobilized trypsin for cleavage of proteins is advantageous in comparison with application of its soluble form. Enzymes can be immobilized on different supports and used in flow systems such as immobilized enzyme reactors (IMERs). This review reports applications of immobilized trypsin reactors in which the IMER has been integrated into separation systems such as reversed-phase liquid chromatography or capillary electrophoresis, prior to MS analysis. Immobilization procedures including supports, mode of integration into separation systems, and methods are described.     

Controllable and high-performance immobilized enzyme reactor: DNA-directed immobilization of multienzyme in polyamidoamine dendrimer-functionalized capillaries

In recent years, CE-integrated immobilized enzyme reactors (IMERs) for single-enzyme immobilization have attracted considerable attention. However, there has been little research on multienzyme immobilization in CE. Here, we introduce a method for fabricating a CE-integrated IMER, using DNA-directed immobilization to fix glucose oxidase and horseradish peroxidase in the capillary, which had been functionalized with polyamidoamine dendrimer (PAMAM). Owing to the reversibility of DNA hybridization, the reactor is capable of dynamic immobilization. Moreover, by introducing the PAMAM, the loading capacity of the IMER is greatly enhanced, and the PAMAM can spontaneously form complexes with DNA and then contribute to the efficiency and stability of the reactor. After 25 days storage, the prepared IMER ultimately retained approximately 70% of its initial activity. We also used the IMER to detect glucose, and the favorable linearity was obtained over the concentration range of 0.78–12.5 mM, with an LOD of 0.39 mM, demonstrating that the CE-integrated IMER can be applied to actual samples. We believe that this strategy can be extended to other multienzyme immobilization systems, and CE-integrated IMERs are potentially useful in a wide range of biochemical research applications.     

Monolith-based immobilized enzyme reactors: recent developments and applications for proteome analysis

In the current proteome study, protein digestion is indispensable before proteins could be identified by MS/MS, no matter based on top-down or bottom-up strategies. Compared to the traditional digestion performed in free solution, immobilized enzyme shows great advantages in digestion speed, stability, and longevity, especially with monolithic materials as the supports. Besides the improved digestion capacity, the immobilized enzyme reactors (IMERs) could be further coupled with the separation and detection systems, enabling high-throughput protein identification. In this paper, the latest advances in the monolith-based IMERs and their applications in proteomic study are briefly summarized. By reviewing these achievements, it could be seen that monilith-based IMERs have very bright future in proteome analysis.     

Immobilized enzyme reactors in proteomics

Fast, efficient characterization of proteins is becoming one of the hottest topics in the bioanalytical community, especially for large-scale proteomic studies. As an attractive approach, protein digestion by enzymes supported on various matrices (referred to as immobilized enzyme reactors, IMERs) has recently attracted much attention.In this article, we present a critical overview of some highly efficient IMERs and related analytical systems. We give major coverage to applications of IMERs in proteomic analysis, including protein-expression profiling, characterization of proteins with post-translational modifications, and protein quantification. We also comment on promising trends for IMERs in proteomics.     

Integration of capillary isoelectric focusing with monolithic immobilized pH gradient,immobilized trypsin microreactor and capillary zone electrophoresis for on‐line protein analysis

An integrated platform consisting of protein separation by CIEF with monolithic immobilized pH gradient (M‐IPG), on‐line digestion by trypsin‐based immobilized enzyme microreactor (trypsin‐IMER), and peptide separation by CZE was established. In such a platform, a tee unit was used not only to connect M‐IPG CIEF column and trypsin‐IMER, but also to supply adjustment buffer to improve the compatibility of protein separation and digestion. Another interface was made by a Teflon tube with a nick to couple IMER and CZE via a short capillary, which was immerged in a centrifuge tube filled with 20 mmol/L glutamic acid, to exchange protein digests buffer and keep electric contact for peptide separation. By such a platform, under the optimal conditions, a mixture of ribonuclease A, myoglobin and BSA was separated into 12 fractions by M‐IPG CIEF, followed by on‐line digestion by trypsin‐IMER and peptide separation by CZE. Many peaks of tryptic peptides, corresponding to different proteins, were observed with high UV signals, indicating the excellent performance of such an integrated system. We hope that the CE‐based on‐line platform developed herein would provide another powerful alternative for an integrated analysis of proteins.     

Disparities between immobilized enzyme and solution based digestion of transferrin with trypsin

Trypsin digestion is a major component of preparing proteins for peptide based identification and quantification by mass spectral (MS) analysis. Surprisingly proteolysis is the slowest part of the proteomics process by an order of magnitude. Numerous recent efforts to reduce protein digestion to a few minutes have centered on the use of an immobilized enzyme reactor (IMER) to minimize both trypsin autolysis and vastly increase the trypsin to protein ratio. A central question in this approach is whether proteolysis with an IMER produces the same peptide cleavage products as derived from solution based digestion. The studies reported here examined this question with transferrin; a model protein of known resistance to trypsin digestion. Results from these studies confirmed that a trypsin‐IMER can in fact digest transferrin in a few minutes; providing tryptic peptides that subsequent to MS analysis allow sequence identification equivalent to solution digestion. Although many of the peptides obtained from these two trypsin digestion systems were identical, many were not. The greatest difference was that the trypsin‐ IMER produces (i) numerous peptides bearing multiple lysine and/or arginine residues and (ii) identical portions of the protein sequence were found in multiple peptides. Most of these peptides were derived from five regions in transferrin. These results were interpreted to mean that proteolysis in the case of transferrin occurred faster than the rate at which buried lysine and arginine residues were unmasked in the five regions providing peptides that were only partially digested.     

Rapid and efficient proteolysis through laser-assisted immobilized enzyme reactors

In this report, laser radiation (808nm) for the first time was employed to enhance the efficiency of proteolysis through immobilized enzyme reactor (IMER). IMER based monolithic support was prepared in the fused-silica capillary via a simple two-step procedure including acryloylation on trypsin surface and in situ aqueous polymerization/immobilization. The feasibility and high efficiency of the laser-assisted IMER were demonstrated by the digestion of bovine serum albumin (BSA), cytochrome c (Cyt-c) and β-casein. The digestion process was achieved in 60s. The peptides were identified by MALDI-TOF-MS, yielding the sequence coverage of 33% for BSA, 73% for Cyt-c and 22% for β-casein. The comparisons between the in-solution digestion and on IMER reaction with/without laser assistance were made. To further confirm its efficiency in proteome analysis, the laser-assisted IMER was also applied to the analysis of one fraction of human serum sample through two-dimensional (2-D) separation of strong anion exchange/reversed-phase liquid chromatography (SAX/RPLC). After a database search, 49 unique peptides corresponding to 5 proteins were identified. The results showed that the laser-assisted IMER provides a promising platform for the high-throughput protein identification.     

Efficient Proteolysis of Glycoprotein Using a Hydrophilic Immobilized Enzyme Reactor Coupled with MALDI-QIT-TOF-MS Detection and μHPLC Analysis

A hydrophilic immobilized enzyme reactor (IMER) containing trypsin was prepared and applied in the proteolysis of glycoproteins. Glycoproteins including horseradish peroxidase, asialofetuin, and fetuin were used to evaluate the performance of the hydrophilic IMER for the glycoprotein digestion. The digested products were detected by matrix-assisted laser desorption/ionization quadruple ion trap time-of-flight mass spectrometry and micro-high-performance liquid chromatography. The hydrophilic IMER showed higher enzymatic digestion efficiency compared with conventional in-solution digestion. The digestion time could be reduced from 16 h to several minutes. Furthermore, using microwaves as a heat source, the reproducibility of the hydrophilic IMER was evaluated and this IMER could be recycled for at least ten times without obvious loss of enzyme activity. The hydrophilic IMER provides a promising tool for high-throughput glycoproteome analysis.     

Immobilization of trypsin onto 1,4-diisothiocyanatobenzene-activated porous glass for microreactor-based peptide mapping by capillary electrophoresis: Effect of calcium ions on the immobilization procedure

The immobilization conditions and kinetic behaviour of trypsin, covalently immobilized via the 1,4-diisothiocyanatobenzene (DITC) linker onto aminopropylated controlled pore glass (CPG) particles, have been evaluated to establish a rapid and efficient protocol for fabrication of an immobilized enzyme microreactor (IMER) for protein hydrolysis and subsequent peptide mapping. Addition of calcium ions to either the immobilization reaction solution or hydrolysis assay was studied for a synthetic substrate. Activity was slightly higher when immobilization was carried out in the presence of Ca²⁺ whereas more enzyme could be immobilized in its absence. A protocol requiring less than 3 h was devised to obtain maximal enzymatic activity with the lowest ratio of soluble trypsin to DITC-CPG particles. The resulting immobilized enzyme was found to retain an acceptable percentage (ca. 35%) of its activity after immobilization. The particles were dry-packed into a capillary to make a microscale IMER. Repeatability, reusability and digestion efficiency of the μIMER were investigated for the substrate β-casein using capillary electrophoretic-based peptide mapping. In initial tests, a single device showed reproducible peptide maps for 21 digestions lasting 2 h each, carried out over a period of 2 months. Complete digestion of β-casein could be achieved in a few minutes (86 s residence time in the μIMER followed by a wash step).     

The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.     

Investigation of micro-immobilised enzyme reactors containing endoglucanases for efficient hydrolysis of cellodextrins and cellulose derivatives

Cellulases hydrolysing the interior parts of cellulose, also called endoglucanases, were immobilised in micro-immobilised enzyme reactors (μIMER) made of porous silicon with the purpose of investigating the use of such μIMERs for hydrolysis of cellodextrins and soluble cellulose derivatives. The endoglucanases Trichoderma reesei Cel 12A (TrCel 12A) and Bacillus agaradhaerens Cel 5A (BaCel 5A) were covalently coupled to the surface of a silicon microchip through Schiff base formation. For characterisation cellohexaose was used as substrate for the immobilised enzymes. The characteristics of the μIMER were investigated by studying the product formation when varying the concentration, flow-rate, temperature and pH of the substrate solution. Hydrolysis was performed in the μIMER connected on-line to a chromatographic system, where the products were separated and detected using high-performance anion exchange chromatography (HPAEC) coupled to pulsed amperometric detection (PAD). A comparison of the hydrolytic pattern between BaCel 5A and TrCel 12A was carried out and the results show that the two investigated endoglucanases give specific hydrolytic patterns in the products formed that provide important information about the enzymes. The μIMERs are robust and can be employed continuously over a period of at least several days. Moreover, on appropriate storage, no activity loss is seen after 60 days. The ability of the BaCel 5A containing μIMER to perform hydrolysis of derivatised cellulose was also investigated using carboxymethyl cellulose (CMC) as substrate. Separation and detection were carried out using size exclusion chromatography (SEC) with refractive index detection (RI). The results show that the μIMERs are robust and can be employed for on-line hydrolysis of both cellodextrins and derivatised cellulose of high molecular weight.     

Capillary electrophoresis-integrated immobilized enzyme microreactor with graphene oxide as support: Immobilization of negatively charged L-lactate dehydrogenase via hydrophobic interactions

We report the first application of hydrophobic interaction between graphene oxide (GO) and negatively charged enzymes to fabricate CE-integrated immobilized enzyme microreactors (IMERs) by a simple and reliable immobilization procedure based on layer by layer assembly. L -lactate dehydrogenase (L -LDH), which is negatively charged during the enzymatic reaction, is selected as the model enzyme. Various spectroscopic techniques, including SEM, FTIR, and UV-vis are used to characterize the fabricated CE-IMERs, demonstrating the successful immobilization of enzymes on the negatively charged GO layer in the capillary surface. The IMER exhibits excellent repeatability with RSDs of inter-day and batch-to-batch less than 3.49 and 6.37%, respectively, and the activity of immobilized enzymes remains about 90% after five-day usage. The measured K_m values of pyruvate and NADH of the immobilized L -LDH are in good agreement with those obtained by free enzymes. The results demonstrate that the hydrophobic interactions and/or π-π stacking is significant between the GO backbone and the aromatic residues of L -LDH and favorable to fabrication of CE-integrated IMERs. Finally, the method is successfully applied to the determination of pyruvate in beer samples.     

Immobilized trypsin on epoxy organic monoliths with modulated hydrophilicity: novel bioreactors useful for protein analysis by liquid chromatography coupled to tandem mass spectrometry

The development of epoxy organic monoliths with modulated hydrophilicity for the preparation of novel trypsin-based microreactors is reported. Porous polymer monoliths have been prepared using methacrylate chemistry triggered by γ-ray irradiation. In situ polymerization has been optimized and extended to medium and high polymer densities using glycidyl methacrylate (GMA) as reactive monomer as well as to the hydrophilic nature of the co-monomers (glyceryl monomethacrylate, GlyMA and acrylamide, AMD). Enzyme immobilization was smoothly achieved by passing a buffered trypsin solution through the columns kept at room temperature. The activities of the immobilized enzyme were characterized by the apparent Michaelis constant (K(m)) and the apparent maximum velocity (V(max)) of the reaction using a non chromogenic, low-molecular mass substrate N-α-benzoyl-l-arginine ethyl ester (BAEE). For the kinetic constants determination a new off-line chromatographic procedure was developed on purpose. The most efficient IMERs were obtained by immobilizing trypsin on monolithic skeleton prepared with hydrophilic monomers (GlyMA and AMD). One of the most promising bioreactor was applied to the digestion of model proteins with different molecular weight and complexity such as human serum albumin (HSA), β-casein and ribonuclease B (RNase B), and the produced peptides were analyzed by liquid chromatography-mass spectrometry. Using a digestion time of only 25 min the proteins were recognized by the database with satisfactory sequence coverage, which was 78.22, 49.76 and 80.68% for HSA, β-casein and RNase B, respectively.     

Monolithic micro-immobilized-enzyme reactor with human recombinant acetylcholinesterase for on-line inhibition studies

The development and characterization of a human recombinant acetylcholinesterase (hrAChE) micro-immobilized-enzyme reactor (IMER), prepared by using an in situ immobilization procedure is reported. hrAChE was covalently immobilized on an ethylenediamine (EDA) monolithic convective interaction media (CIM) disk (12 mm x 3 mm i.d.), previously derivatized with glutaraldehyde. The optimal conditions for the immobilization were: 12 microg of enzyme dissolved in 800 microl of phosphate buffer (50 mM, pH 6.0). The mixture was gently agitated overnight at 4 degrees C. The resulting Schiff bases were reduced by cyanoborohydride and the remaining aldehydic groups were condensed with monoethanolamine. Under these conditions, 0.22 U of hrAChE were immobilized with retention of 3.0% of the initial enzymatic activity. The activity of the immobilized hrAChE was stable for over 60 days. The activity and kinetic parameters of the hrAChE micro-IMER were investigated by inserting the micro-IMER in a HPLC system and it was demonstrated that the enzyme retained its activity. The micro-IMER was characterized in terms of units of immobilized enzyme and best conditions for immobilization yield. IMERs were compared for their relative enzyme stability, immobilized units, yield and aspecific matrix interactions. The effect of AChE inhibitors was evaluated by the simultaneous injection of each inhibitor with the substrate. The relative IC50 values were found in agreement with those derived by the conventional kinetic spectrophotometric method. In comparison with previously developed AChE-based IMERs, AChE monolithic micro-IMER showed advantages in terms of reduction of analysis time (2 min), lower aspecific matrix interactions and lower backpressure. Included in a HPLC system, it can be used for the rapid screening of new compounds' inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analyzed in continuous.     

An improved design to capture magnetic microparticles for capillary electrophoresis based immobilized microenzyme reactors

In this paper, we demonstrate the effectiveness of a new 3D printed magnet holder that enables capture of magnetic microparticles in commercially available capillary electrophoresis equipment with a liquid or air based coolant system. The design as well as the method to capture magnetic microparticles inside the capillary are discussed. This setup was tested at temperature and pH values suitable for performing enzymatic reactions. To demonstrate its applicability in CE‐ immobilized microenzyme reactors (IMER) development, human flavin‐containing monooxygenase 3 and bovine serum albumin were immobilized on amino functionalized magnetic microparticles using glutaraldehyde. These microparticles were subsequently used to perform in‐line capillary electrophoresis with clozapine as a model substrate. This setup could be used further to establish CE‐IMERs of other drug metabolic enzymes in a commercially available liquid based capillary coolant system. The CE‐IMER setup was successful, although a subsequent decrease in enzyme activity was observed on repeated runs.     

Rheostatic control of tryptic digestion in a microscale fluidic system

Integrated fluidic systems that unite bottom-up and top-down proteomic approaches have the potential to deliver complete protein characterization. To circumvent fraction collection, as is conducted in current blended approaches, a technique to regulate digestion efficiency in a flow-through system is required. The present study examined the concept of regulating tryptic digestion in an immobilized enzyme reactor (IMER), incorporating mixed solvent systems for digestion acceleration. Using ovalbumin, cytochrome c, and myoglobin as protein standards, we demonstrate that tryptic digestion can be efficiently regulated between complete digestion and no digestion extremes by oscillating between 45 and 0% acetonitrile in the fluid stream. Solvent composition was tuned using programmable solvent waveforms in a closed system consisting of the IMER, a sample delivery stream, a dual gradient pumping system and a mass spectrometer. Operation in this rheostatic digestion mode provides access to novel peptide mass maps (due to substrate unfolding hysteresis) as well as the intact protein, in a reproducible and stable fashion. Although cycle times were on the order of 90 s for testing purposes, we show that regulated digestion is sufficiently rapid to be limited by solvent switching efficiency and kinetics of substrate unfolding/folding. Thus, regulated digestion should be useful in blending bottom-up and top-down proteomics in a single closed fluidic system.     

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K = 2.65 × 10⁻¹³ m²). With BSA as the model protein, efficient digestion was completed within 20 s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12 h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20 s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.