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.     

Integrated protein analysis platform based on column switch recycling size exclusion chromatography,microenzymatic reactor and μRPLC–ESI-MS/MS

An integrated platform with the combination of proteins and peptides separation was established via the unit of on-line proteins digestion, by which proteins were in sequence separated by column switch recycling size exclusion chromatography (csrSEC), on-line digested by an immobilized trypsin microreactor, trapped and desalted by two parallel C8 precolumns, separated by μRPLC with the linear gradient of organic modifier concentration, and identified by ESI-MS/MS. A 6-protein mixture, with Mr ranging from 10 kDa to 80 kDa, was used to evaluate the performance of the integrated platform, and all proteins were identified with sequence coverage over 5.67%. Our experimental results demonstrate that such an integrated platform is of advantages such as good time compatibility, high peak capacity, and facile automation, which might be a promising approach for proteome study.     

Nano-flow multidimensional liquid chromatography platform integrated with combination of protein and peptide separation for proteome analysis

An integrated multidimensional nano-flow liquid chromatography platform with the combination of protein and peptide separation via online digestion by an immobilized enzymatic reactor was established, and successfully applied for proteome analysis. By this platform, proteins were first separated by a weak anion and weak cation mixed-bed microcolumn under a series of salt steps, online digested by a trypsin immobilized enzymatic reactor, digests trapped and desalted by a C18 precolumn, separated by nano-reversed phase liquid chromatography, and finally identified by electrospray ionization-MS/MS. To evaluate the performance of such a platform, Escherichia coli whole cell lysate proteins were analyzed. Compared with the results obtained by shotgun approach, the identified protein number was increased by 6%, with the consumed time decreased from 38 to 14 h. We also compared with integrate platform based on micro-HPLC, and the required sample amount was decreased to 8 μg. These results demonstrated that such an integrated approach would be an attractive alternative to commonly applied approaches for proteome research.     

集成化蛋白质组定量分析平台的构建及应用

为提高蛋白质组定量分析的准确度、通量和自动化程度,构建了由微升级混合离子交换色谱、亲水型固定化酶反应器（hIMER）和纳升级反相色谱-电喷雾串级质谱（nanoRPLC-ESI-MS/MS）组成的集成化蛋白质定量分析平台。该平台实现了二甲基化标记蛋白质样品在线分离、酶解、肽段分离鉴定和定量分析。采用质量比为1：1的轻、重标记的蛋白质样品考察该平台的定量性能,发现蛋白质水平二甲基化标记效率为90%;蛋白质经hIMER在线酶解10 min产生的漏切及酶解产物在hIMER柱上的非特异性吸附对定量准确度的影响较小,所有定量到的重/轻标记的蛋白质质量比的平均值为1.01。最后将该平台应用于小鼠腹水型肝癌淋巴道高、低转移细胞系差异蛋白质的分析,发现了12种蛋白质在高转移细胞系中低表达,15种蛋白质在高转移细胞系中高表达。以上结果证明了该平台可以实现高准确度和高通量的蛋白质组定量分析。     

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.     

On-line protein digestion and peptide mapping by capillary electrophoresis with post-column labeling for laser-induced fluorescence detection

A nanoliter enzyme microreactor was developed for on-line capillary electrophoresis (CE) peptide mapping of proteins, allowing picomole quantities of proteins to be digested. The enzyme microreactor was formed by immobilizing trypsin onto a monolithic capillary column, which was prepared by in situ polymerization of glycidyl methacrylate and ethylene dimethacrylate in a capillary. Highly efficient digestion of three protein standards was demonstrated. The detection of peptide fragments in CE was enhanced by post-column derivatization and laser-induced fluorescence detection. The microreactor has a volume of about 30 nL and is coupled with a separation capillary via a fluid joint for on-line digestion. The overall analysis, including digestion and separation, lasted only about 16 min. Column efficiencies > 300 000 plates/m were obtained for most peaks in the electropherogram of an on-line peptide mapping experiment of denatured alpha-lactalbumin under optimal conditions.     

Ion-exchange-membrane-based enzyme micro-reactor coupled online with liquid chromatography-mass spectrometry for protein analysis

In this article, we developed a membrane-based enzyme micro-reactor by directly using commercial polystyrene–divinylbenzene cation–exchange membrane as the support for trypsin immobilization via electrostatic and hydrophobic interactions and successfully applied it for protein digestion. The construction of the reactor can be simply achieved by continuously pumping trypsin solution through the reactor for only 2 min, which was much faster than the other enzyme immobilization methods. In addition, the membrane reactor could be rapidly regenerated within 35 min, resulting in a “new” reactor for the digestion of every protein sample, completely eliminating the cross-interference of different protein samples. The amount and the activity of immobilized trypsin were measured, and the repeatability of the reactor was tested, with an RSD of 3.2% for the sequence coverage of cytochrome c in ten digestion replicates. An integrated platform for protein analysis, including online protein digestion and peptide separation and detection, was established by coupling the membrane enzyme reactor with liquid chromatography–quadrupole time-of-flight mass spectrometry. The performance of the platform was evaluated using cytochrome c, myoglobin, and bovine serum albumin, showing that even in the short digestion time of several seconds the obtained sequence coverages was comparable to or higher than that with in-solution digestion. The system was also successfully used for the analysis of proteins from yeast cell lysate.     

Combining capillary electrophoresis with mass spectrometry for applications in proteomics

Mass spectrometry (MS)-based proteomics is currently dominated by the analysis of peptides originating either from digestion of proteins separated by two-dimensional gel electrophoresis (2-DE) or from global digestion; the simple peptide mixtures obtained from digestion of gel-separated proteins do not usually require further separation, while the complex peptide mixtures obtained by global digestion are most frequently separated by chromatographic techniques. Capillary electrophoresis (CE) provides alternatives to 2-DE for protein separation and alternatives to chromatography for peptide separation. This review attempts to elucidate how the most promising CE modes, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), might best be applied to MS-based proteomics. CE-MS interfacing, mass analyzer performance, column coating to minimize analyte adsorption, and sample stacking for CZE are considered prior to examining numerous applications. Finally, multidimensional systems that incorporate CE techniques are examined; CZE often finds use as a fast, final dimension before ionization for MS, while CIEF, being an equilibrium technique, is well-suited to being the first dimension in automated fractionation systems.     

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.     

复合纤维素膜固定化胰蛋白酶反应器及其应用于蛋白质酶解

合成了甲基丙烯酸缩水甘油酯-纤维素复合膜,并以此膜为基质共价键合固定化胰蛋白酶,以N-苯甲酰-L-精氨酰乙酯(BAEE)为底物,应用高效液相色谱系统测定了酶固定化膜柱的催化反应特性。研究结果表明:温度、pH值、离子强度、有机溶剂及蛋白变性剂等都对固定化酶的活力有一定的影响。在最适条件下,固定化胰蛋白酶的活力为17800U/g干膜,蛋白载量为3.6mg/g(≈0.15μmol/g)干膜,活性回收率达到52%.固定化酶表现出较高的使用和储藏稳定性,在40℃下,水解BAEE底物24h活力无显着变化。固定化酶膜柱在4℃冷藏保存100d仍保存90%以上的水解活力。固定化酶反应器被应用于蛋白质酶解的肽谱实验。     

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

In the presence of methanol and n‐decanol as porogens, a partially filled capillary monolithic column was prepared by in situ reaction of glycidyl methacrylate and poly (ethylene glycol) diacrylate. Then, Pharmalyte 3–10 was immobilized on this column in order to obtain a capillary isoelectric focusing (cIEF) column with monolithic immobilized pH gradient (M‐IPG). In addition, an online self‐built platform for protein separation was established on account of the introduction of a cross‐shaped unit and two short‐off valves. In this platform, a cross‐shaped unit was not only used to join the M‐IPG column and a six‐way injection valve (1.5 μL sample loop), but also to supply a volume pool of anode buffer so that the process of injection, focusing and mobilization of samples could be sequentially performed. The short‐off valve in the tee unit or cross‐shaped unit could be used to control the direction of the fluid flow. Using this online cIEF platform and under the optimized conditions, 7‐proteins mixture could be separated and a good linear correlation between pI values and migration times was obtained by the M‐IPG column. Meanwhile, based on the online cIEF platform, human serum proteins and a mixture of Hb A and Hb A_1c have been successfully resolved with the newly developed M‐IPG column.     

Efficient protein digestion with peptide separation in a micro-device interfaced to electrospray mass spectrometry

A highly efficient protein digestion device has been fabricated using commercially available immobilized trypsin on agarose beads, packed into a silica capillary and connected either directly to an electrospray mass spectrometer via a 'microtight T' connector, from which aqueous acetic acid (0.2%) was pumped, or via a monolithic column connected to the mass spectrometer ion source. Six proteins with molecular mass ranging from 2848 to 77703 Da were digested completely using this system. In the second set of experiments a short monolithic separation column was placed after the immobilized trypsin capillary and partial separation of the generated peptides was obtained. The detection limits were increased from the micromol to pmol range by utilization of this separation column. Gradient elution, using a binary HPLC pump and a flow splitter, was used to optimize the peptide separation. This provided significantly enhanced resolution of the tryptic peptides but increased the analysis time to 30 minutes.     

Trypsin entrapped in poly(diallyldimethylammonium chloride) silica sol-gel microreactor coupled to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

An enzyme-immobilized capillary microreactor for rapid protein digestion and proteomics analysis is reported. The inner surface of the fused-silica capillary was coated with poly(diallyldimethylammonium chloride) (PDDA)-entrapped silica sol-gel matrix, followed by assembly of trypsin onto the PDDA-modified surface via electrostatic adsorption. The immobilization parameters such as PDDA content in the sol-gel matrix, trypsin concentration and pH were investigated in detail. Protein samples including beta-casein, myoglobin and cytochrome c could be effectively digested and electrophoretically separated simultaneously in such a modified capillary. Just 2.26 ng (corresponding to 0.10-0.14 picomole) of sample was sufficient for on-line capillary electrophoresis peptide mapping. The efficiency of the digestion was further demonstrated by digestion of a human liver cytoplasm sample and 253 proteins were identified in one unique run.     

Trypsin inhibitor screening in traditional Chinese medicine by using an immobilized enzyme microreactor in capillary and molecular docking study

A trypsin immobilized enzyme microreactor was successfully prepared in capillary for studying enzyme kinetics of trypsin and online screening of trypsin inhibitors from traditional Chinese medicine through capillary electrophoresis. Trypsin was immobilized on the inner wall at the inlet of the capillary treated with polydopamine. The rest of the capillary was used as a separation channel. The parameters including the separation efficiency and the activity of immobilized trypsin were comprehensively evaluated. Under the optimal conditions, online screening of trypsin inhibitors each time can be carried out within 6 min. The Michaelis–Menten constant of immobilized trypsin was calculated to be 0.50 mM, which indicated high affinity of the immobilized trypsin for the substrate. The half‐maximal inhibitory concentration of known inhibitor of benzamidine hydrochloride hydrate as a model inhibitor was 13.32 mM. The proposed method was successfully applied to screen trypsin inhibitors from 15 compounds of traditional Chinese medicine. It has been found that baicalin showed inhibitory potency. Molecular docking study well supported the experimental result by exhibiting molecular interaction between enzyme and inhibitors.     

固定化胰蛋白酶整体小柱的制备及其用于微量蛋白质的快速酶解

发展了一种光引发聚合法制备固定化胰蛋白酶整体小柱的方法,以用于微量蛋白质的快速酶解。整体小柱由功能单体4-戊烯酸琥珀酰亚胺酯、甲基丙烯酸羟乙酯,交联剂季戊四醇三丙烯酸酯和三元致孔剂二甲基亚砜、N,N-二甲基甲酰胺、十二醇在20 μL的移液器吸头尖端原位聚合而成。形成整体柱后,胰蛋白酶分子通过氨基与琥珀酰亚胺酯反应实现固定化。系统研究了聚合溶液中活性酯含量与柱床体积对胰蛋白酶固载量的影响,评价了固定化酶整体小柱对标准蛋白细胞色素C和牛血清白蛋白的酶解效率,以及整体小柱的稳定性和重复性。结果表明,在离心辅助下,酶解过程可在10 min内完成,批次间具有良好的重复性。最后将固定化酶整体小柱应用于1×10⁵个人急性早幼粒白血病（NB4）细胞与人急性T细胞白血病（Jurkat T）细胞的快速酶解,经纳升级液相色谱与高分辨质谱联用分析后鉴定得到2489个和2572个蛋白质。相比于溶液状态下的酶解,分别提高了2.2%和6.1%的蛋白鉴定数量,展现了其在蛋白组学研究中的应用潜力。     

Development of an on-line immobilized-enzyme reversed-phase HPLC method for protein digestion and peptide separation

This paper describes use of a novel glass bead-based immobilized-enzyme micro column for simple and swift on-line protein digestion then peptide separation by reversed-phase HPLC. The inexpensive and easily made immobilized-enzyme micro column was prepared from aminopropyl controlled-pore glass that was reacted first with glutaraldehyde then with trypsin in the presence of phosphate buffer. Tryptic digestion of bovine serum albumin (BSA) was achieved simply by passing pretreated protein solution through the laboratory made immobilized-trypsin column; the tryptic fragments were then separated by reversed-phase HPLC. The peptide separation was found to be identical to separation of a sample which had undergone conventional enzymatic protein digestion in solution. Digestion of BSA by the immobilized-trypsin column decreased with increasing flow rate of the solution through the column, and 1.0 μL min⁻¹ was found to be the optimum flow rate for on-line protein digestion with our system. It was also found that the sample required pretreatment with urea before injection, because of a change in the properties of the protein in the presence of urea, and the immobilized-trypsin column lost its function in the presence of acetonitrile. This on-line proteomics system enables simple and rapid protein digestion and was successfully applied to partially micro two-dimensional (2D) chromatographic separation of proteins.     

Poly(glycidyl methacrylate-divinylbenzene) based immobilized pH gradient capillary isoelectric focusing coupling with MALDI mass spectrometry for enhanced neuropeptide analysis

Herein, we report an immobilized pH gradient (IPG) capillary isoelectric focusing-matrix-assisted laser desorption/ionization mass spectrometry (CIEF-MALDI MS) platform designed for the separation of complex neuropeptides. This platform features a poly(glycidyl methacrylate-divinylbenzene) (GMA-DVB)-based monolithic column for CIEF separation. Different from regular CIEF, carrier ampholytes are preimmobilized on the monolithic surface instead of being added to the sample. An off-line coupling of IPG-CIEF to MALDI MS has been established. Comparison with regular CIEF and optimizations are performed with bovine serum albumin tryptic peptides and extracted neuropeptide mixtures from crustacean Callinectes sapidus. It has been demonstrated that the separation of complex peptide mixtures in neutral and basic pH ranges can be achieved in less than 10 min with comparable separation efficiency with regular CIEF, while the MS signal is significantly enhanced when employing IPG-CIEF. Enhanced neuropeptide detection is also observed after coupling IPG-CIEF with MALDI MS.     

基于核酸适配体的微流控芯片酶反应器用于蛋白质的分析

将核酸适配体作为胰蛋白酶固定化介质,制备了一种新型的微流控芯片酶反应器,并与高效液相色谱-串联质谱联用,搭建了在线分析平台;分别使用标准蛋白及混合蛋白样品对芯片的酶解效率及联用平台的分析能力进行了初步评价。结果表明,5 ng肌红蛋白经该平台分析后肽段覆盖率可达到37%;对500 ng混合蛋白进行3次平行分析,肽段覆盖率及相对标准偏差分别为44.3%、6.5%(牛血清白蛋白), 65.0%、2.7%(肌红蛋白)和62.0%、5.6%(细胞色素c);初步实验表明,该在线分析平台具有检测灵敏度高、重现性好、酶解效率高的特点,有望在蛋白质组学分析中发挥重要作用。     

Application of a temperature-controllable microreactor to simple and rapid protein identification using MALDI-TOF MS

We have carried out a simultaneous thermal denaturation and trypsin digestion of proteins using a temperature-controllable microreactor. This is a simple and rapid sample preparation technique for use before matrix-assisted laser desorption ionization time-of-flight mass spectrometry. In contrast to a conventional sample preparation method, which involves several chemical treatments, our sample preparation was performed using only trypsin digestion with the thermal denaturation of the target protein. Optimization of the reactor operational parameters for trypsin digestion using a temperature-controllable microreactor was carried out. The entire trypsin digestion procedure took about 11 min, and consisted of 1 min for the thermal denaturation of the sample protein (3 microl, 0.2 microM) at 85 degrees C, and 10 min for digestion of the protein at 37 degrees C. The resulting sequence coverage ranged from 24% to 57%, which was sufficient for practical protein identification.