Immobilized Enzyme Reactor Research Articles

Electrochemical Measurements of Glucose Using a Micro Flow‐Through Immobilized Enzyme Reactor

AbstractA new fast, cheap and efficient epoxy‐amine immobilized enzyme reactor (IMER) is demonstrated. Polyethyleneimine (PEI) was used as the curing agent of an epoxy resin (bisphenol‐A diglycidyl ether (BADGE)) in order to introduce a high number of reactive –NH2 groups at the substrate. A ratio mixture of 3:2 PEI:BADGE (w/w) was shown to be the most suitable for curing, taking into account the number of immobilization sites and the resistance of the material towards channeling. Electrochemical measurements made by a three electrode system, adapted at the IMER, showed that the Km value for immobilized glucose oxidase (GOx) was half the value commonly reported for GOx immobilized at PEI derived substrates. The IMER response decreased by around 41 % after one week of intense usage. Even so, the remaining activity was sufficient for quantifications of approximately 0.1 mmol L−1, merely requiring a new calibration of the reactor before its utilization. The developed system was applied to D‐glucose quantification of beverage samples, requiring an injection volume of only 10 μL and a flow rate of 150 μL min−1 (almost 60 samples per hour). Low detection and quantification limits (1.94 and 5.89 μmol L−1, respectively) and a wide linear range (from 8 μmol L−1 to 2 mmol L−1) were also found, which are useful characteristics for quality control analysis.

Taylor dispersion analysis, resonant mass measurement and bioactivity of pepsin-coated gold nanoparticles

Immobilized enzyme reactors based on nanoparticulate carriers are becoming increasingly popular. A toolbox of methods is usually utilized for their characterization in order to be capable of assessing their suitability for the intended purpose. In this work, as a model system pepsin was conjugated to gold nanoparticles (GNPs) by a straightforward adsorptive immobilization process. The success of the immobilization procedure was monitored by Vis spectroscopy via shifts of the localized surface plasmon resonance (LSPR) band, size characterization by dynamic light scattering (DLS), and ζ–potential determinations by electrophoretic light scattering. DLS revealed a significantly different hydrodynamic diameter for unmodified GNPs and protein-coated GNPs. However, the hydrodynamic diameters of pepsin-coated GNPs obtained with various concentrations of pepsin in the coating solution were not significantly different. In contrast, Taylor dispersion analysis allowed measuring the slight differences in the hydrodynamic radius. It provided also information on the viscosity of GNP suspensions and diffusion coefficients for the various pepsin@GNP preparations. For the determination of the pepsin surface coverage on the GNPs results from indirect protein quantitation of non-immobilized pepsin by Lowry assay were compared to direct measurement of immobilized pepsin by resonant mass measurements. Reasonable agreement was found. Accurate information on enzyme coverage is of utmost importance for a representative comparison of the turnover numbers and catalytic efficiencies of nanoparticulate immobilized enzyme reactors, which is shown by the adsorptively immobilized pepsin@GNP as model system.

Towards automation in protein digestion: Development of a monolithic trypsin immobilized reactor for highly efficient on-line digestion and analysis

Reducing experimental variability, limiting contamination and increasing automation are essential goals in the development of reliable analytical platforms for mass spectrometry (MS)-based proteomics. In this work novel trypsin-based monolithic immobilized enzyme reactors (tryp-IMERs), obtained by covalent immobilization on convective interaction media (CIMac™) analytical columns (5mm×5.2mm I.D.), were developed. Notwithstanding the small dimensions, column format allowed the insertion in common high performance liquid chromatography (HPLC) systems, thus avoiding the use of expensive micro- or nano-platforms. Monolith pore diameter and surface chemistry were optimized to achieve high digestion efficiency even with high molecular weight proteins and to avoid protein/peptide adsorption, peak broadening and sample loss. A full characterization of the tryp-IMERs was undertaken to select the best protocol for preparation and type of trypsin. Optimization of the operational and storage conditions was carried out by an off-line approach. On-line studies were performed by setting a multidimensional analytical platform, which included the tryp-IMER, a trapping column, an analytical C4 column and a high resolution hybrid mass spectrometer (ESI-Q-TOF). In the optimized conditions rapid protein digestion (90±9s), high protein coverage (≥60%) and high score values were achieved for five selected sample proteins (cytochrome c, myoglobin and albumins from different sources) differing in molecular size, isoelectric point and accessibility to cleavage sites as well as for a protein mixture of 200ng. The best performing tryp-IMERs showed high sensitivity down to the pmole level. The platform also resulted suitable for the analysis of high-molecular weight proteins such as a pool of human immunoglobulins G (hIgG) and for the high molecular weight fraction of human plasma proteins, which were digested in less than two minutes to an extent similar to that achieved by overnight incubation in a classical in solution protocol. Finally, underestimated key procedural issues were also highlighted during the study. Such aspects are of general interest both for tryp-IMER users and tryp-IMER developers.

A cyclic-olefin-copolymer microfluidic immobilized-enzyme reactor for rapid digestion of proteins from dried blood spots

A critical step in the bottom-up characterization of proteomes is the conversion of proteins to peptides, by means of endoprotease digestion. Nowadays this method typically uses overnight digestion and as such represents a considerable bottleneck for high-throughput analysis. This report describes protein digestion using an immobilized-enzyme reactor (IMER), which enables accelerated digestion times that are completed within seconds to minutes. For rapid digestion to occur, a cyclic-olefin-copolymer microfluidic reactor was constructed containing trypsin immobilized on a polymer monolithic material through a 2-vinyl-4,4-dimethylazlactone linker. The IMER was applied for the rapid offline digestion of both singular protein standards and a complex protein mixture prior to liquid chromatography–electrospray ionisation-tandem mass spectrometry (LC–ESI-MS/MS) analysis. The effects of protein concentration and residence time in the IMER were assessed for protein standards of varying molecular weight between 11 and 240kDa. Compared to traditional in-solution digestion, IMER-facilitated protein digestion at room temperature for 5min yielded similar results in terms of sequence coverage and number of identified peptides. Good repeatability was demonstrated with a relative standard deviation of 6% for protein-sequence coverage. The potential of the IMER was also demonstrated for a complex protein mixture in the analysis of dried blood spots. Compared to a traditional workflow a similar number of proteins could be identified, while reducing the total analysis time from 22.5h to 4h and importantly omitting the sample-pre-treatment steps (denaturation, reduction, and alkylation). The identified proteins from two workflows showed similar distributions in terms of molecular weight and hydrophobic character.

Preparation and characterization of a packed bead immobilized trypsin reactor integrated into a PDMS microfluidic chip for rapid protein digestion

This paper demonstrates the design, efficiency and applicability of a simple, inexpensive and high sample throughput microchip immobilized enzymatic reactor (IMER) for rapid protein digestion. The IMER contains conventional silica particles with covalently immobilized trypsin packed inside of a poly(dimethylsiloxane) (PDMS) microchip channel (10mm×1mm×35µm). The microchip consists of 9 different channels, enabling 9 simultaneous protein digestions. Trypsin was covalently immobilized using carbodiimide activation, the ideal trypsin/silica particle ratio (i.e. measured mass ratio before the immobilization reaction) was determined. The amount of immobilized trypsin was 10–15μg trypsin for 1mg silica particle. Migration times of CZE peptide maps showed good repeatability and reproducibility (RSD%=0.02–0.31%). The IMER maintained its activity for 2 months, in this period it was used effectively for rapid proteolysis. Four proteins (myoglobin, lysozyme, hemoglobin and albumin) in a wide size range (15–70kDa) were digested to demonstrate the applicability of the reactor. Their CZE peptide maps were compared to peptide maps obtained from standard in-solution digestion of the four proteins. The number of peptide peaks correlated well with the theoretically expected peptide number in both cases, the peak patterns of the electropherograms were similar, however, digestion with the microchip IMER requires only <10s, while in-solution digestion takes 16h. LC-MS/MS peptide mapping was also carried out, the four proteins were identified with satisfying sequence coverages (29–50%), trypsin autolysis peptides were not detected. The protein content of human serum was digested with the IMER and with in-solution digestion.

Achieving efficient digestion faster with Flash Digest: potential alternative to multi-step detergent assisted in-solution digestion in quantitative proteomics experiments.

In quantitative analysis of protein biomarkers and therapeutic proteins by liquid chromatography/mass spectrometry (LC/MS), it is a preferred and well-established approach to digest with proteolytic enzymes to produce smaller peptide fragments which are more suitable for LC/MS analysis than the intact protein. In-solution digestion is one widely used method for protein digestion. Proteolytically resistant proteins often require digestion times that extend beyond normal working hours and prohibit same day analysis. We evaluated the performance of an immobilized enzyme reactor (IMER) to determine if this technology could reduce method development time, digestion time and increase throughput. We digested human plasma samples using a commercially available IMER, Flash Digest, and compared it to an in-solution digestion method for analysis of three different apolipoprotein biomarkers APOE, APOC2, and APOC3. The plasma digests were analyzed via LC/MS using electrospray ionization (ESI) and multiple reaction monitoring (MRM). Value assigned calibrators were selected over a relevant physiological concentration range for each protein of interest. Quality control samples (QCs) and 'unknown' human plasma samples were analyzed with both methods. Flash Digest significantly reduced digestion time for APOC3, the most proteolytically resistant of the three proteins, to 30min compared with overnight used with in-solution digestion. The Flash Digest achieved comparable digestion efficiency with minimal method development and reduced sample preparation time. Both methods showed linearity over a physiologically relevant concentration range. Precision was evaluated and a percentage coefficient of variance (% CV) less than 8% was obtained during intra-day reproducibility evaluation for all three apolipoproteins with Flash Digest. Concentrations observed for QCs and unknown samples using Flash Digest were comparable to the in-solution method. An IMER such as Flash Digest may be a potential alternative to in-solution digestion to accelerate digestion of proteolytically resistant proteins in a quantitative proteomics experiments, reduce method development time and increase throughput. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of an immobilized enzyme reactor for on-line protein digestion

Despite the developments for faster liquid chromatographic and mass spectral detection techniques, the standard in-solution protein digestion for proteomic analyses has remained relatively unchanged. The typical in-solution trypsin protein digestion is usually the slowest part of the workflow, albeit one of the most important. The development of a highly efficient immobilized enzyme reactor (IMER) with rapid performance for on-line protein digestion would greatly decrease the analysis time involved in a proteomic workflow. Presented here is the development of a silica based IMER for on-line protein digestion, which produced rapid digestions in the presence of organic mobile phase for both model proteins and a complex sample consisting of the insoluble portion of a yeast cell lysate. Protein sequence coverage and identifications evaluated between the IMER and in-solution digestions were comparable. Overall, for a yeast cell lysate with only a 10s volumetric residence time on-column, the IMER identified 507 proteins while the in-solution digestion identified 490. There were no significant differences observed based on identified protein’s molecular weight or isoelectric point between the two digestion methods. Implementation of the IMER into the proteomic workflow provided similar protein identification results, automation for sample analysis, and reduced the analysis time by 15h.

On-column trypsin digestion coupled with LC-MS/MS for quantification of apolipoproteins

Apolipoproteins measured in plasma or serum are potential biomarkers for assessing metabolic irregularities that are associated with the development of cardiovascular disease (CVD). LC-MS/MS allows quantitative measurement of multiple apolipoproteins in the same sample run. However, the accuracy and precision of the LC-MS/MS measurement depends on the reproducibility of the enzymatic protein digestion step. With the application of an immobilized enzyme reactor (IMER), the reproducibility of the trypsin digestion can be controlled with high precision via flow rate, column volume and temperature. In this report, we demonstrate the application of an integrated IMER-LC-MS/MS platform for the simultaneous quantitative analysis of eight apolipoproteins. Using a dilution series of a characterized serum pool as calibrator, the method was validated by repeated analysis of pooled sera and individual serum samples with a wide range of lipid profiles, all showing intra-assay CV<4.4% and inter-assay CV<8%. In addition, the method was compared with traditional homogeneous digestion coupled LC-MS/MS for the quantification of apoA-I and apoB-100. Applied in large scale human population studies, this method can serve the translation of a wider panel of apolipoprotein biomarkers from research to clinical application. SignificanceCurrently, the translation of apolipoprotein biomarkers to clinical application is impaired because of the high cost of large cohort studies using traditional single-analyte immunoassays. The application of on-line tryptic digestion coupled with LC-MS/MS analysis is an effective way to address this problem. In this work we demonstrate a high throughput, multiplexed, automated proteomics workflow for the simultaneous analysis of multiple proteins.

Capillary electrophoresis-integrated immobilized enzyme reactors

Abstract Online enzyme assay based on capillary electrophoresis (CE) offers several advantages for the assay, such as low consumption of samples, easy automation of all steps, and less requirement of sample work-up. As a widely used approach for online enzyme assay, CE-integrated immobilized enzyme microreactor (IMER) has been applied in almost all aspects of enzyme assays during the past two decades, including evaluation of the enzymatic activity and kinetics, screening of inhibitor, investigation of enzyme-mediated metabolic pathways, and proteome analysis. In a CE-integrated IMER, enzyme is bound to the capillary surface or a suitable carrier attached to the capillary and substrates/products of the enzymatic reaction are separated and online detected by CE at downstream of the capillary. Enzymatic reactions can be viewed as interaction between the stationary phase (immobilized enzyme) and the mobile phase (substrate(s)/co-enzyme(s) solution), in analogy to the well-known separation technique, capillary electrochromatography. From this point of view, CE-integrated IMERs can be categorized into open tubular capillary IMER, monolithic IMER, and packed capillary IMER. In this review, we have surveyed, analyzed, and discussed advances on fabrication techniques of the three categories of CE-integrated IMERs for online assays involving various enzymes in the past two decades (1992–2015). Some recent studies using microfluidic-based IMERs for enzyme assays have also been reviewed.

Preparation of graphene oxide-modified affinity capillary monoliths based on three types of amino donor for chiral separation and proteolysis

Novel graphene oxide (GO)-modified affinity capillary monoliths were developed employing human serum albumin (HSA) or pepsin as chiral selector. Three types of amino donors for GO immobilization, including ammonium hydroxide (NH4OH), ethanediamine (EDA) and polyethyleneimine (PEI), were applied to explore the effect of spacer arm on enantioseparation. It was observed that HSA-GO-EDA-based affinity capillary monoliths exhibited better chiral recognition ability in comparison with the other two spacer-based monoliths. Under the optimized conditions, the obtained columns revealed satisfactory repeatability concerning column-to-column, run-to-run and interday repeatability. In addition, the impact of GO concentration on enantiomeric separation was also investigated. HSA-GO-EDA-based affinity capillary monoliths provided higher chiral selectivity for nine pairs of enantiomers compared to the columns without GO. Furthermore, the influence of amino donors and GO on proteolytic activity of pepsin-based immobilized enzymatic reactor (IMER) was discussed. Unfortunately, pepsin-GO-PEI-based affinity capillary monoliths possessed the highest protein digestion capacity, which was different from the effect of amino donors on enantiorecognition. Moreover, GO presented as a favorable choice to improve the enzymatic activity of IMER. These results proved that GO-functionalized affinity capillary monoliths have promising potential for chiral separation and proteolysis.

Characterization of Pharmaceutical IgG and Biosimilars Using Miniaturized Platforms and LC-MS/MS.

Therapeutic monoclonal antibodies (mAbs) have made a tremendous impact in treating patients with various diseases. MAbs are designed to specifically target a cell and illicit a response from the immune system to destroy the cell. As originator mAb drug patents are coming to an end, generic pharmaceutical companies are poised to replicate and produce so-called biosimilar drugs. MAbs are significantly more complicated than small drugs to analyze and produce. The mAb proteoform and glycoform must be as similar to the original drug as possible to be a viable replacement. The mAb proteoform is well characterized but can be altered through various undesirable reactions such as deamidation. The mAb glycoform is harder to replicate as the glycan formation is a complicated templateless one; it is proving difficult for the originator companies to produce a homogenous population of mAbs from batch to batch. Severe side-effects have occurred in patients taking mAbs with immunogenic glycans, highlighting the importance of quality control mechanisms. The complex nature of mAbs requires sensitive and robust tools amenable to the highthroughput analysis required by a manufacturing setting. Miniaturized analytical platforms for complex biosimilar analysis are still in their infancy but have shown great promise for sample preparation. Capillary electrophoresis-laser induced fluorescence remains a powerful and fast technique for routine glycan analysis. Mass spectrometry is the method of choice for the analysis of mAb proteoforms and is emerging as a powerful tool for glycoform analysis.

Protein-Labs on Separative Analytical Scale in Medicinal Chemistry: from the Proof of Concept to Applications

Coupling of a macromolecule such as an enzyme or a receptor to a chromatographic support has revealed a powerful analytical tool to face different aspects related to drug discovery. Chromatographic systems with immobilized macromolecules have been developed to be used for different applications in pharmaceutical analysis including enzyme inhibitor screening, biopharmaceutics structural analysis, chiral separations and binding studies. These areas of research are very challenging both for the technological and for the applicative aspects. This review offers an overview of general strategies and references that can be considered in order to develop innovative and reliable “protein-labs” for specific separative analytical applications of pharmaceutical interest. Keywords: Protein immobilization, bio-affinity chromatography, immobilized enzyme reactors, chiral stationary phases, drug binding, enantioseparation, inhibitor potency determination.

A facile and efficient method of enzyme immobilization on silica particles via Michael acceptor film coatings: immobilized catalase in a plug flow reactor.

A novel method was developed for facile immobilization of enzymes on silica surfaces. Herein, we describe a single-step strategy for generating of reactive double bonds capable of Michael addition on the surfaces of silica particles. This method was based on reactive thin film generation on the surfaces by heating of impregnated self-curable polymer, alpha-morpholine substituted poly(vinyl methyl ketone) p(VMK). The generated double bonds were demonstrated to be an efficient way for rapid incorporation of enzymes via Michael addition. Catalase was used as model enzyme in order to test the effect of immobilization methodology by the reactive film surface through Michael addition reaction. Finally, a plug flow type immobilized enzyme reactor was employed to estimate decomposition rate of hydrogen peroxide. The highly stable enzyme reactor could operate continuously for 120h at 30°C with only a loss of about 36% of its initial activity.

Application of BACE1 immobilized enzyme reactor for the characterization of multifunctional alkaloids from Corydalis cava (Fumariaceae) as Alzheimer's disease targets

In our ongoing study focused on Corydalis cava (Fumariaceae), used in folk medicine in the treatment of memory dysfunctions, we have investigated fifteen previously isolated alkaloids for their potential multifunctional activity on Alzheimer's disease (AD) targets. Determination of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibition was carried out using a BACE1-Immobilized Enzyme Reactor (IMER) by validating the assay with a multi-well plate format Fluorescence Resonance Energy Transfer (FRET) assay. Seven alkaloids out of fifteen were found to be active, with (−)-corycavamine (3) and (+)-corynoline (5) demonstrating the highest BACE1 inhibition activity, in the micromolar range, in a concentration dependent manner. BACE1-IMER was found to be a valid device for the fast screening of inhibitors and the determination of their potency. In a permeation assay (PAMPA) for the prediction of blood–brain barrier (BBB) penetration, the most active compounds, (−)-corycavamine (3) and (+)-corynoline (5), were found to be able to cross the BBB. Not all compounds showed activity against glycogen synthase kinase-3β (GSK-3β) and casein kinase-1δ (CK-1δ). On the basis of the reported results, we found that some C. cava alkaloids have multifunctional activity against AD targets (prolyl oligopeptidase, cholinesterases and BACE1). Moreover, we tried to elucidate the treatment effectivity (rational use) of its extract in memory dysfunction in folk medicine.

Immobilization of NTPDase-1 from Trypanosoma cruzi and Development of an Online Label-Free Assay.

The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems is an interesting approach in the screening of new ligands. In addition, IMERs offer many advantages over techniques that employ enzymes in solution. The enzyme nucleoside triphosphate diphosphohydrolase (NTPDase-1) from Trypanosoma cruzi acts as a pathogen infection facilitator, so it is a good target in the search for inhibitors. In this paper, immobilization of NTPDase-1 afforded ICERs (Immobilized Capillary Enzyme Reactors). A liquid chromatography method was developed and validated to monitor the ICER activity. The conditions for the application of these bioreactors were investigated, and excellent results were obtained. The enzyme was successfully immobilized, as attested by the catalytic activity detected in the TcNTPDase-1-ICER chromatographic system. Kinetic studies on the substrate ATP gave K M of 0.317 ± 0.044 mmol·L−1, which still presented high affinity compared to in solution. Besides that, the ICER was stable for 32 days, enough time to investigate samples of possible inhibitors, including especially the compound Suramin, that inhibited 51% the enzyme activity at 100 µmol·L−1, which is in accordance with the data for the enzyme in solution.

Recent advances in CE mediated microanalysis for enzymatic and derivatization reactions.

This review gives an overview of the applications and recent advances in CE mediated microanalysis. As a continuation of earlier reviews, it covers articles published from a period of mid 2013-early 2015. The article contains three main parts. In the first part of the article, different kinds of in-line CE mediated microanalysis are briefly discussed along with relevant papers regarding in-line CE for enzyme analysis. Recent advances in on-capillary derivatization reactions and immobilized enzyme reactors (IMERs) have been summarized along with their purpose and relevance in the second and third part of the paper, respectively.

Low back-pressure hierarchically structured multichannel microfluidic bioreactors for rapid protein digestion – Proof of concept

A novel, easy-to-fabricate monolithic enzymatic microreactor with a hierarchical, torturous structure of flow-through channels of micrometric sizes and large mesopores was shown to enable rapid and very efficient digestion of proteins at high yields and exceptionally low back-pressures. Four silica monoliths with bi-modal 3D pore structure in micrometer and nanometer size scales were synthesized and characterized for structural and flow properties. The monolith with the highest total pore volume (4cm3/g) and flow-through channels 20–30μm in size, was further functionalized with trypsin to obtain multichannel immobilized enzyme (proteolytic) reactor (IMER). The value of permeability coefficient K evaluated for water (∼2.0·10−11) was found to be two orders of magnitude higher in the novel reactor than reported before for high-performance IMERs, enabling the flow rates of 750mL/cm2min at pressure gradients of 64kPa/cm. Very high practical potentials of the novel microbioreactor were demonstrated in the proteolysis of cytochrome c (Cyt-c) and myoglobin (Myo), without any earlier pretreatment. MALDI-TOF/TOF mass spectrometry analysis of sequence coverage was high: 70% (Cyt-c) and 90% (Myo) for 24min digestion, and 39% (Cyt-c) and 53% (Myo) when the proteolysis time was reduced to 2.4min. The proposed microreactors make full use of all advantages of microfuidic devices and mesoporous biocatalysts, and offer exceptional possibilities for biochemical/proteolytic applications in both large (production) and small (analytical) scales.

Hyphenation of a Deoxyribonuclease I immobilized enzyme reactor with liquid chromatography for the online stability evaluation of oligonucleotides

The stability of antisense oligonucleotides (ONs) toward nucleases is a key aspect for their possible implementation as therapeutic agents. Typically, ON stability studies are performed off-line, where the ONs are incubated with nucleases in solution, followed by their analysis. The problematics of off-line processing render the detailed comparison of relative ON stability quite challenging. Therefore, the development of an online platform based on an immobilized enzyme reactor (IMER) coupled to liquid chromatography (LC) was developed as an alternative for improved ON stability testing. More in detail, Deoxyribonuclease I (DNase I) was immobilized on epoxy-silica particles of different pore sizes and packed into a column for the construction of an IMER. Subsequently, the hyphenation of the IMER with ion-pair chromatography (IPC) and ion-exchange chromatography (IEC) was evaluated, leading to the successful development of two online methodologies: IMER-IPC and IMER-IEC. More specifically, natural and modified DNA and RNA oligonucleotides were used for testing the performance of the methodologies. Both methodologies proved to be simple, automatable, fast and highly reproducible for the quantitative and qualitative evaluation of ON degradation. In addition, the extended IMER life time in combination with a more straightforward control of the reaction kinetics substantiate the applicability of the IMER-LC platform for ON stability tests and its implementation in routine and research laboratories.

A one-step preparation method of monolithic enzyme reactor for highly efficient sample preparation coupled to mass spectrometry-based proteomics studies

Mass spectrometry (MS) coupled to sample preparation and separation techniques has become a primary tool for proteomics studies. However, due to sample complexity, it is often challenging to achieve fast and efficient sample preparation prior to MS analysis. In recent decades, monolithic materials have been developed not only as chromatographic media, but also as efficient solid supports for immobilizing multiple types of affinity reagents. Herein, the N-acryloxysuccinimide-co-acrylamide-co-N,N′-methylenebisacrylamide (NAS-AAm-Bis) monolith was fabricated within silanized 200μm i.d. fused-silica capillaries and was used as an immobilized enzyme reactor (IMER). The column was conjugated with trypsin/Lys-C and Lys-N enzymes to allow enzymatic digestions to occur while protein mixture was loaded onto the IMER column followed by MS-based proteomics analysis. Similar MS signal and protein sequence coverage were observed using protein standard bovine serum albumin (BSA) compared to in-solution digestion. Furthermore, mouse serum, yeast, and human cell lysate samples were also subjected to enzymatic digestion by both IMER (in seconds to minutes) and conventional in solution digestion (overnight) for comparison in large-scale proteomics studies. Comparable protein identification results obtained by the two methods highlighted the potential of employing NAS-based IMER column for fast and highly efficient sample preparation for MS analysis in proteomics studies.

Flow‐Synthesis of Nucleosides Catalyzed by an Immobilized Purine Nucleoside Phosphorylase from Aeromonas hydrophila: Integrated Systems of Reaction Control and Product Purification

AbstractA purine nucleoside phosphorylase from Aeromonas hydrophyla (AhPNP) was covalently immobilized in a pre‐packed stainless steel column containing aminopropylsilica particles via Schiff base chemistry upon glutaraldehyde activation. The resulting AhPNP‐IMER (Immobilized Enzyme Reactor, immobilization yield ≈50%) was coupled on‐line through a 6‐way switching valve to an HPLC apparatus containing an analytical or a semi‐preparative chromatographic column. The synthesis of five 6‐modified purine ribonucleosides was carried out by continuously pumping the reaction mixture through the AhPNP‐IMER until the highest conversion was reached, and then directing the reaction mixture to chromatographic separation. The conditions of the AhPNP‐catalyzed transglycosylations (2:1 ratio sugar donor:base acceptor; 10 mM phosphate buffer; pH 7.5; temperature 37 °C, flow rate 0.5 mL min−1) were optimized by a fractional factorial experimental design. Coupling the bioconversion step with the product purification in such an integrated platform resulted in a fast and efficient synthetic process (yield=52–89%; &lt;10 mg) where sample handling was minimized. To date, AhPNP‐IMER has retained completely its activity upon 50 reactions in 10 months.magnified image