Miniaturized on-line proteolysis-capillary liquid chromatography-mass spectrometry for peptide mapping of lactate dehydrogenase

In this study, methodology was developed for on-line and miniaturized enzymatic digestion with liquid chromatographic (LC) separation and mass spectrometric (MS) detection. A packed capillary LC-MS system was combined with on-line trypsin cleavage of a model protein, lactate dehydrogenase, to provide an efficient system for peptide mapping. The protein was injected onto an enzymatic capillary reactor and the resulting peptides were efficiently trapped on a capillary trapping column. Different trapping columns were evaluated to achieve a high binding capacity for the peptides generated in the enzyme reactor. The peptides were further eluted from the pre-column and separated on an analytical capillary column by a buffer more suitable for the following an electrospray ionisation (ESI) MS process. An important aspect of the on-line approach was the desalting of peptides performed in the trapping column to avoid detrimental signal suppression in the ESI process. The developed on-line system was finally compared to a classical digestion in solution, with reference to peptide sequence coverage and sensitivity. It was shown that the on-line system gave more than 100% higher peptide sequence coverage than traditional digestion methods.     

A replaceable microreactor for on-line protein digestion in a two-dimensional capillary electrophoresis system with tandem mass spectrometry detection

We describe a two-dimensional capillary electrophoresis system that incorporates a replaceable enzymatic microreactor for on-line protein digestion. In this system, trypsin is immobilized on magnetic beads. At the start of each experiment, old beads are flushed to waste and replaced with a fresh plug of beads, which is captured by a pair of magnets at the distal tip of the first capillary. For analysis, proteins are separated in the first capillary. A fraction is then parked in the reactor to create peptides. Digested peptides are periodically transferred to the second capillary for separation; a fresh protein fraction is simultaneously moved to the reactor for digestion. An electrospray interface is used to introduce peptides into a mass spectrometer for analysis. This procedure is repeated for several dozen fractions under computer control. The system was demonstrated by the separation and digestion of insulin chain b oxidized and β-casein as model proteins.     

Optimization of flow-injection systems for determination of substrates by means of enzyme amplification reactions and chemiluminescence detection

A flow-injection system is described that incorporates a small column reactor containing two co-immobilized, synergistically operating oxidoreductases, allowing determination of minute amounts of substrates by means of enzyme amplification and subsequent chemiluminescence detection of the hydrogen peroxide generated in the repeated redox cycling. With lactate oxidase and lactate dehydrogenase, and taking advantage of the fact that the enzymatic degradation step and the ensuing detection step can be individually optimized, the FIA-system has been optimized by factorial experiments to yield an amplification factor of over 140 for each of the two substrates lactate and pyruvate. With a linear calibration range of 0-6muM, the limits of detection for the two species were 48 and 103nM, respectively, and the sampling rate was 50-60/hr. The optimized system has also been employed for assay of glucose by utilizing a column reactor with immobilized glucose oxidase and glucose dehydrogenase, but yielded amplification factors of only 3-4. The large discrepancy in the performance of the two enzyme systems is discussed.     

Post-column reactor of coaxial-gap mode for laser-induced fluorescence detection in capillary electrophoresis

A post-column reactor with coaxial-gap mode is developed for laser-induced fluorescence detection (LIF) in capillary electrophoresis (CE). The reactor can be assembled simply and conveniently, in which a thin polyimide sleeve of 10-mm length obtained from the capillary coating is used to align separation and reaction capillary with a 20 microm gap. Naphthalene-2,3-dicarboxaldehyde and 2-mercaptoethanol are used as derivatization reagents and delivered into the reaction capillary through the annulus between the separation capillary and polyimide sleeve and the gap of two capillaries by gravity. A reaction distance from the gap to detection point is 5mm. For the post-column reactor of CE-LIF, several configuration parameters are optimized, including liquid level difference between the derivatization solution and outlet buffer, annular dimension between the outer diameter of etched separation capillary and the inner diameter of polyimide sleeve, and reaction distance, etc. The detection limits in the range from 8.0x10(-8) to 1.0x10(-6) mol/L and linear calibration range more than two orders of magnitude are obtained for amino acids. The separation efficiency ranges from 1.35x10(5) to 1.67x10(5) theoretical plates.     

Microchip-based capillary electrophoresis for determination of lactate dehydrogenase isoenzymes

This article describes a novel microchip-based capillary electrophoresis and oncolumn enzymatic reaction analysis protocol for lactate dehydrogenase (LDH) isoenzymes with a home-made xenon lamp-induced fluorescence detection system. A microchip integrated with a temperature-control unit is designed and fabricated for low-temperature electrophoretic separation of LDH isoenzymes, optimal enzyme reaction temperature control, and product detection. A four-step operation and temperature control are employed for the determination of LDH activity by on-chip monitoring of the amount of incubation product of NADH during the fixed incubation period and at a fixed temperature. Experiments on the determination of LDH standard sample and serum LDH isoenzymes from a healthy adult donor are carried out. The results are comparable with those obtained by conventional CE. Shorter analysis times and a more stable and lower background baseline can be achieved. The efficient separation of different LDH forms indicates the potential of microfluidic devices for isoenzyme assay.     

A chemiluminometric method for NADPH and NADH using a two-enzyme bioreactor and its application to the determination of magnesium in serum

Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. When the sample is introduced into the four-enzyme bioreactor, hydrogen peroxide is produced in proportion to the concentration of serum magnesium by the successive reactions. A co-immobilized hexokinase/glucose-6-phosphate dehydrogenase/glutamate dehydrogenase column reactor gave better efficiency compared with an enzyme column which was prepared by packing co-immobilized hexokinase/glucose-6-phosphate dehydrogenase and immobilized glutamate dehydrogenase to make two layers. Magnesium in serum was determined with 1 microL of the sample without carry-over and for an assay time of approximately 15 s. The present method is sensitive (detection limit 0.1 nmol) because Mg2+ is recycled in a column, and gives perfect linearity of the data up to 3.0 mmol/L with satisfactory precision, reproducibility, and accurate reaction recoveries.     

Advances in Capillary Electrophoresis-Based Enzyme Assays

Capillary electrophoresis (CE) has become a flexible and accurate, high-efficiency analytical separation technique in many areas requiring only minute amounts of sample and chemicals. Thus, CE has also been recognized as a suitable technique to study enzymatic reactions including the determination of Michaelis–Menten kinetic data or the identification and characterization of inhibitors. The most often applied CE-based enzyme assay modes can be divided into two categories: (1) pre-capillary assays where incubations are performed offline followed by CE analysis of substrate(s) and/or product(s) and (2) in-capillary assays in which the enzymatic reaction and analyte separation are performed in the same capillary. In case of the in-capillary assays, the enzyme may be immobilized or in solution. The latter is also referred to as electrophoretically mediated microanalysis (EMMA), while in the case of immobilized enzyme the term immobilized enzyme reactor (IMER) is used. The present review summarizes the literature on CE-based enzyme assays published between January 2010 and April 2015. Immobilized enzyme reactors as well as microfluidic devices applied to the study of enzymatic activity will also be briefly addressed.

     

Off-line and in-line determination of catechol- O-methyltransferase activity in a capillary electrophoretic system

The use of capillary electrophoresis for the determination of catechol-O-methyltransferase (COMT) activity with dihydroxybenzoic acid as a substrate was investigated. Both an off-line and in-line capillary electrophoresis determination of COMT activity was developed and the two approaches are discussed. In the presented methods, substrate and reaction products are monitored at the same time. The initial velocity of the reaction is quantified spectrophotometrically by the corrected peak area of the products at 200 nm. In the off-line setup, capillary zone electrophoresis is used to separate and quantify the different reaction compounds. Each electrophoretic run required only 37 nL of the enzymatic reaction solution. Based on the off-line assay, an in-line determination of COMT activity was developed by a methodology known as electrophoretically mediated microanalysis (EMMA). All the different steps (i.e. mixing, incubation, separation and in-line quantitation) are combined in the capillary, which is used as a microreactor for the enzymatic reaction. Full automation of the assay is achieved with this microscale approach.     

毛细管区带电泳用于酶微反应器酶解条件的研究

将氨基酰化酶通过戊二醛固定在毛细管内壁,制备毛细管酶微反应器,用毛细管区带电泳对毛细管酶微反应器的酶解产物进行分离,以生成物的峰面积优化底物N-乙酰-DL-蛋氨酸的酶解条件。实验结果表明,在温度37℃的条件下,10μg/mL N-乙酰-DL-蛋氨酸磷酸盐缓冲溶液(pH7.5)以4μL/min的速度通过15 cm长的毛细管酶微反应器,具有良好的酶解效果。利用毛细管酶微反应器对底物N-乙酰-DL-蛋氨酸进行酶解,每天酶解5次,10天后酶活仅下降了8.66%,说明制备的毛细管酶微反应器具有良好的稳定性。     

Microchip CE‐LIF method for the hydrolysis of L‐glutamine by using L‐asparaginase enzyme reactor based on gold nanoparticle

l ‐Asparaginase (l ‐Asnase) can suppress the growth of malignant cells by rapid depletion of two essential amino acids, l ‐glutamine (l ‐Gln) and l ‐asparagine (l ‐Asn). To study the cytotoxic effect and the secondary complications of l ‐Asnase in the treatment of acute lymphoblastic leukemia, the development of a novel enzyme reactor of l ‐Asnase for the hydrolysis of l ‐Gln, employing the enzyme‐gold nanoparticle conjugates in capillary, was reported in this work. First, a microchip CE (MCE)‐LIF was established for the separation of l ‐amino acids (l ‐Gln and l ‐glutamic acid) and studying the hydrolysis of l ‐Gln by using l ‐Asnase enzyme reactor. Then, using l ‐Gln as target analyte, the enzyme kinetics of l ‐Asnase in free solution, enzyme‐gold nanoparticle conjugates (E‐GNP), and the enzyme‐gold nanoparticle conjugates immobilized in capillary (E‐GNP‐C) were investigated in detail with the proposed MCE‐LIF method. Moreover, for optimizing the enzymatic reaction efficiency, three important parameters, including the length of capillary, the enzyme concentration reacted with gold nanoparticle and the amount of l ‐Asnase immobilized on the gold nanoparticle, have been studied. Owing to the high specific activity, the E‐GNP‐C enzyme reactor exhibited the best performance for the hydrolysis of l ‐Gln.     

毛细管电泳序列分析技术用于在线酶分析研究进展

毛细管电泳技术具有操作简单、样品消耗量少、分离效率高和分析速度快等优势,不仅是一种高效的分离分析技术,而且已经发展成为在线酶分析和酶抑制研究的强有力工具。酶反应全程的实时在线监测,可以实现酶反应动力学过程的高时间分辨精确检测,以更准确地获得反应机制和反应速率常数,有助于更好地了解酶反应机制,从而更全面深入地认识酶在生物代谢中的功能。此外,准确、快速的在线酶抑制剂高通量筛选方法的发展,对加快酶抑制类药物的研发以及疾病的临床诊断亦具有重要意义。电泳媒介微分析法（EMMA）和固定化酶微反应器（IMER）是毛细管电泳酶分析技术中常用的在线分析方法。这两种在线酶分析法的进样方式通常为流体动力学进样和电动进样,无法实现酶反应过程中的无干扰序列进样分析。近年来,基于快速序列进样的毛细管电泳序列分析技术已经发展成为在线酶分析的另一种强有力手段,以实现高时间分辨和高通量的酶分析在线检测。该文从快速序列进样的角度,综述了近年来毛细管电泳序列分析技术在线酶分析的研究进展,并着重介绍了各种序列进样方法及其在酶反应和酶抑制反应中的应用,包括光快门进样、流动门进样、毛细管对接的二维扩散进样、流动注射进样、液滴微流控进样等。     

Determination of glycerol, 1,2-propanediol and triglycerides by high-performance liquid chromatography and a post-column reactor containing immobilized glycerol dehydrogenase

A fluorimetri method is described for the determination of glycerol, 1,2-propanediol and triglycerides in serum by high-performance liquid chromatography with an on-line post-column reactor containing immobilized glycerol dehydrogenase. Before separation, triglycerides are cleaved with lipase and esterase. The polyhydric alcohols are separated from each other on a Finepak SIL C₁₈ (10 μm) column with water as eluent. The NADHI produced from the enzymatic reaction is monitored by fluorimetry. Calibration curves are linear between 0.01 mM and 1.0 mM for glycerol or 2.0 mM for 1,2-propanediol. The method gave satisfactory results for control sera.     

Asymmetric reduction of acetophenone in membrane reactors: comparison of oxazaborolidine and alcohol dehydrogenase catalysed processes

The enantioselective reduction of acetophenone was studied in two different ways. Chemical borane reduction using a homogeneously soluble polymer-bound oxazaborolidine catalyst was carried out in a continuously operated membrane reactor and yielded (R)-phenylethanol in good enantiomeric excess with high space–time yields. An enzymatic reduction using a dehydrogenase two-enzyme system as the catalyst and formate as the hydrogen source was carried out in an extractive bi-membrane reactor and yielded (S)-phenylethanol in excellent enantiomeric excess with a low enzyme consumption. A comparison of the two systems with respect to space–time yield, total turnover number and other parameters is made.     

In‐capillary reactant mixing for monitoring glycerol kinase kinetics by CE

CE was used for the first time to study the two‐substrate enzyme glycerol kinase. The capillary was used as a nanoreactor in which the enzyme and its two substrates glycerol and adenosine‐5′‐triphosphate were in‐capillary mixed to realize the enzymatic assay. For kinetic parameters determination, reactants were injected (50 mbar × 5 s) as follows: (i) incubation buffer; (ii) adenosine‐5′‐triphosphate; (iii) enzyme, and (iv) glycerol. Enzymatic reaction was then initiated by mixing the reactants using electrophoretically mediated microanalysis (+20 kV for 6 s) followed by a zero‐potential amplification step of 3 min. Finally, electrophoretic separation was performed; the product adenosine‐5′‐diphosphate was detected at 254 nm and quantified. For enzyme inhibition, an allosteric inhibitor fructose‐1,6‐bisphosphate plug was injected before the first substrate plug and +20 kV for 8 s was applied for reactant mixing. A simple, economic, and robust CE method was developed for monitoring glycerol kinase activity and inhibition. Only a few tens of nanoliters of reactants were used. The results compared well with those reported in literature. This study indicates, for the first time, that at least four reactant plugs can be in‐capillary mixed using an electrophoretically mediated microanalysis approach.     

Specific detection of nicotinamide coenzymes by liquid chromatography and amperometric detection with immobilized glucose-6-phosphate dehydrogenase

A liquid chromatographic system for the specific and simultaneous detection of nicotinamide coenzymes is constructed by combining an immobilized glucose-6-phosphate dehydrogenase reactor with an amperometric system based on a phenazine methosulphate-mediated reaction, after separation on a reversed-phase column. The calibration graphs are linear from 0.05 to 20 nmol for all four coenzymes. The detection limits are 3.2, 5.2, 7.9 and 9.4 pmol for NADP⁺, NADPH, NAD⁺ and NADH, respectively. The enzyme reactor retains most of its original activity after repeated use for 2 months.     

Determination of aliphatic amino acids in serum by HPLC with fluorimetric detection using co-immobilized enzyme reactor

A simple and selective method is presented for the determination of aliphatic amino acids such as L-alanine, L-valine, L-isoleucine and L-leucine in serum using HPLC with detection by co-immobilized alanine dehydrogenase/leucine dehydrogenase post-column reactor and fluorimeter. The enzymes were simultaneously immobilized on chitosan beads. The separation was achieved by means of an ods column with elution with phosphate buffer (pH 7.0). The system gave linear responses over two orders of magnitude and detection limits at 1-2muM levels.     

Development of off-line and on-line capillary electrophoresis methods for the screening and characterization of adenosine kinase inhibitors and substrates

Fast and convenient CE assays were developed for the screening of adenosine kinase (AK) inhibitors and substrates. In the first method, the enzymatic reaction was performed in a test tube and the samples were subsequently injected into the capillary by pressure and detected by their UV absorbance at 260 nm. An MEKC method using borate buffer (pH 9.5) containing 100 mM SDS (method A) was suitable for separating alternative substrates (nucleosides). For the CE determination of AMP formed as a product of the AK reaction, a phosphate buffer (pH 7.5 or 8.5) was used and a constant current (95 microA) was applied (method B). The methods employing a fused-silica capillary and normal polarity mode provided good resolution of substrates and products of the enzymatic reaction and a short analysis time of less than 10 min. To further optimize and miniaturize the AK assays, the enzymatic reaction was performed directly in the capillary, prior to separation and quantitation of the product employing electrophoretically mediated microanalysis (EMMA, method C). After hydrodynamic injection of a plug of reaction buffer (20 mM Tris-HCl, 0.2 mM MgCl2, pH 7.4), followed by a plug containing the enzyme, and subsequent injection of a plug of reaction buffer containing 1 mM ATP, 100 microM adenosine, and 20 microM UMP as an internal standard (I.S.), as well as various concentrations of an inhibitor, the reaction was initiated by the application of 5 kV separation voltage (negative polarity) for 0.20 min to let the plugs interpenetrate. The voltage was turned off for 5 min (zero-potential amplification) and again turned on at a constant current of -60 microA to elute the products within 7 min. The method employing a polyacrylamide-coated capillary of 20 cm effective length and reverse polarity mode provided good resolution of substrates and products. Dose-response curves and calculated K(i) values for standard antagonists obtained by CE were in excellent agreement with data obtained by the standard radioactive assay.     

Capillary electrophoresis integrated immobilized enzyme reactor for kinetic and inhibition assays of β‐secretase as the Alzheimer's disease drug target

Capillary electrophoresis integrated immobilized enzyme reactors are becoming an increasingly popular alternative for enzyme kinetic and inhibition assays thanks to their unique set of features including cost effectiveness, repeated use of the enzyme, minuscule sample consumption, rapid analysis time and easy automation. In this work we present the development and application of a capillary electrophoresis integrated immobilized enzyme reactor based on magnetic particles for kinetic and inhibition studies of β‐secretase, a key enzyme in the development of Alzheimer's disease and a promising drug target. We document the optimization of the immobilization procedure, characterization of immobilized β‐secretase, optimization of a mutually compatible incubation protocol and separation method as well as the production of the capillary electrophoresis integrated immobilized enzyme reactor. The applicability of the capillary electrophoresis integrated immobilized enzyme reactor was demonstrated by kinetic assay with an unlabelled substrate and by inhibition assays using three structurally different reference inhibitors. The resulting kinetic and inhibition parameters clearly support the applicability of the herein presented method as well as document the fundamental phenomena which need to be taken in account when comparing the results to other methods.     

Determination of l-alanine in a flow-injection system with an immobilized enzyme reactor

A flow-injection system for the determination of l-alanine is described. Alanine dehydrogenase is immobilized on poly(vinyl alcohol) beads and used in a packed-bed enzyme reactor. The system responds linearly to injected samples (50 μl) in the concentration range 0.5–500 μM. The maximum throughput was 40 samples per hour. The immobilized enzyme reactor was stable for at least 6 weeks. Its usefulness for assay of l-alanine in serum and beverages is described.     

超高速平板通道毛细管电泳

超高速平板通道毛细管电泳是９０年代发展的一种秒级分离的新颖技术。应用现代微电子光刻技术将化学反应。进样、分离和检测等组合在数厘米玻片上。实现分离分析的小型化、集成化、一体化和自动化。