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。     

Integrated platform of capillary isoelectric focusing, trypsin immobilized enzyme microreactor and nanoreversed-phase liquid chromatography with mass spectrometry for online protein profiling

An integrated platform with the combination of protein and peptide separation was established via online protein digestion, by which proteins were first separated by CIEF, online digested by a trypsin immobilized enzyme microreactor, trapped and desalted by two parallel trap columns, separated by nanoreversed-phase and finally identified by MS. In such a platform, two hollow fiber membrane interfaces were used. One was applied to supply catholyte and electric contact, and another to supply adjustment buffer to improve the compatibility of protein separation and tryptic digestion. A poly(octadecyl acrylate-co-ethylene dimethacrylate) monolithic column served as the trap column to capture sample and to remove the ampholytes from CIEF. A hybrid silica monolith-based immobilized trypsin microreactor was used for online protein digestion. To evaluate the performance of such a platform, a 4-protein mixture with a loading amount of only 0.29?μg, was analyzed, and sequence coverages for BSA, myoglobin, β-lactoglobulin and ribonuclease A were 8, 26, 10 and 54%, respectively. Furthermore, such an integrated platform was successfully applied for the analysis of proteins extracted from Escherichia coli, and 101 proteins were positively identified. We anticipate that the integrated platform developed herein will provide a promising tool for low-abundance protein identification with the combination of top-down and bottom-up approaches.     

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).     

Vinyl functionalized silica hybrid monolith-based trypsin microreactor for on line digestion and separation via thiol-ene "click" strategy

A novel thiol-ene "click" strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic-inorganic monolithic capillary column with ene-functionality was fabricated by sol-gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα-p-tosyl-L-arginine methyl ester hydrochloride, TAME) and the product (Nα-p-tosyl-L-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N'-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene "click" strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.     

On-line characterization of the activity and reaction kinetics of immobilized enzyme by high-performance frontal analysis

A microreactor by immobilized trypsin on the activated glycidyl methacrylate-modified cellulose membrane packed column was constructed. Immobilized trypsin mirrored the properties of the free enzyme and showed high stability. A novel method to characterize the activity and reaction kinetics of the immobilized enzyme has been developed based on the frontal analysis of enzymatic reaction products, which was performed by the on-line monitoring of the absorption at 410 nm of p-nitroaniline from the hydrolysis of N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA). The hydrolytic activity of the immobilized enzyme was 55.6% of free trypsin. The apparent Michaelis-Menten kinetics constant (Km) and Vmax values measured by the frontal analysis method were, respectively, 0.12 mM and 0.079 mM min(-1) mg enzyme(-1). The former is very close to that observed by the static and off-line detection methods, but the latter is about 15% higher than that of the static method. Inhibition of the immobilized trypsin by addition of benzamidine into substrate solution has been studied by the frontal analysis method. The apparent Michaelis-Menten constant of BAPNA (Km), the inhibition constant of benzamidine (Ki) and Vmax were determined. It was indicated that the interaction of BAPNA and benzamidine with trypsin is competitive, the Km value was affected but the Vmax was unaffected by the benzamidine concentration.     

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.     

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.     

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.     

Analytical characterization of a facile porous polymer monolithic trypsin microreactor enabling peptide mass mapping using mass spectrometry

A simple and rapid single-step method is presented to fabricate an enzyme reactor using trypsin immobilized on a macroporous polymer monolith. A reactor produced in a capillary format is ready to use within 1 h of preparation. The monomers making up the monolith, including N-acryloxysuccinimide for covalent immobilization of the enzyme, are mixed with trypsin and introduced into the column by capillary force for polymerization/immobilization. The enzyme activity from column-to-column is reproducible below 5% relative standard deviation (RSD), while the reactor is durable for at least 20 weeks when stored at room temperature. The apparent kinetic constants V(max) and K(m) are of value similar to those obtained by free trypsin in solution. Enzymatic digestion of proteins was shown to be feasible on a time-scale of seconds and submicromolar concentrations enabling peptide mass mapping by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.     

Immobilized metal-ion chelating capillary microreactor for peptide mapping analysis of proteins by matrix assisted laser desorption/ ionization-time of flight-mass spectrometry

Peptide mass mapping analysis, utilizing a regenerable enzyme microreactor with metal-ion chelated adsorption of enzyme, combined with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) was developed. Different procedures from the conventional approaches were adopted to immobilize the chelator onto the silica supports, that is, the metal chelating agent of iminodiacetic acid (IDA) was reacted with glycidoxypropyltrimethoxysilane (GLYMO) before its immobilization onto the inner wall of the fused-silica capillary pretreated with NH(4)HF(2). The metal ion of copper and subsequently enzyme was specifically adsorbed onto the surface to form the immobilized enzyme capillary microreactor, which was combined with MALDI-TOF-MS to apply for the mass mapping analysis of nL amounts of protein samples. The results revealed that the peptide mapping could routinely be generated from 0.5 pmol protein sample in 15 min at 50 degrees C, even 20 fmol cytochrome c could be well digested and detected.     

Comparison of two glutaraldehyde immobilization techniques for solid-phase tryptic peptide mapping of human hemoglobin by capillary zone electrophoresis and mass spectrometry

Stabilization of proteolytic enzymes, especially by immobilization, is of considerable interest because of their potential applications in medicine and the chemical and pharmaceutical industries. We report here a detailed comparison of two procedures for trypsin immobilization using the same homobifunctional agent, glutaraldehyde, for the purpose of peptide mapping. These methods include covalent coupling either to controlled pore glass (solid support) or via a cross-linking reaction (without any solid support). The immobilized trypsin preparations were characterized by the determination of immobilization efficiency, which ranged from 68 to > 95%, and measurement of apparent kinetic parameters toward a synthetic peptide-like substrate. Batch digestions of whole denaturated human normal adult hemoglobin (HbA) were performed to obtain peptide maps by capillary zone electrophoresis (CZE). Migration time reproducibility of the CZE maps was excellent, with a mean relative standard deviation of 1.5%. Moreover, the two immobilized enzyme preparations showed excellent reproducibility for repeated digestions. Matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry was also used for peptide mass mapping of denaturated HbA digested using the two immobilized trypsin preparations. Even though the two immobilized trypsin preparations do not behave identically, similar sequence coverages of 57% and 61% (for the two HbA chains merged) were achieved for the support-based and cross-linked trypsin preparations, respectively.     

Evaluation inhibitory activity of catechins on trypsin by capillary electrophoresis–based immobilized enzyme microreactor with chromogenic substrate

In this study, a capillary electrophoresis‐based online immobilized enzyme microreactor was developed for evaluating the inhibitory activity of green tea catechins and tea polyphenol extracts on trypsin. The immobilized trypsin activity and other kinetic parameters were evaluated by measuring the peak area of the hydrolyzate of chromogenic substrate S‐2765. The results indicated that the activity of the immobilized trypsin remained approximately 90.0% of the initial immobilized enzyme activity after 30 runs. The value of Michaelis–Menten constant (K_m) was (0.47 ± 0.08) mM, and the half‐maximal inhibitory concentration (IC₅₀) and inhibition constant (K_i) of benzamidine were measured as 3.34 and 3.00 mM, respectively. Then, the inhibitory activity of four main catechins (epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate) and three tea polyphenol extracts (green tea, white tea, and black tea) on trypsin were investigated. The results showed that four catechins and three tea polyphenol extracts had potential trypsin inhibitory activity. In addition, molecular docking results illustrated that epigallocatechin gallate, epicatechin gallate, epicatechin, and epigallocatechin were all located not only in the catalytic cavity, but also in the substrate‐binding pocket of trypsin. These results indicated that the developed method is an effective tool for evaluating inhibitory activity of catechins on trypsin.     

Improvement of proteolytic efficiency towards low-level proteins by an antifouling surface of alumina gel in a microchannel

A microfluidic reactor has been developed for rapid enhancement of protein digestion by constructing an alumina network within a poly(ethylene terephthalate) (PET) microchannel. Trypsin is stably immobilized in a sol-gel network on the PET channel surface after pretreatment, which produces a protein-resistant interface to reduce memory effects, as characterized by X-ray fluorescence spectrometry and electroosmotic flow. The gel-derived network within a microchannel provides a large surface-to-volume ratio stationary phase for highly efficient proteolysis of proteins existing both at a low level and in complex extracts. The maximum reaction rate of the encapsulated trypsin reactor, measured by kinetic analysis, is much faster than in bulk solution. Due to the microscopic confinement effect, high levels of enzyme entrapment and the biocompatible microenvironment provided by the alumina gel network, the low-level proteins can be efficiently digested using such a microreactor within a very short residence time of a few seconds. The on-chip microreactor is further applied to the identification of a mixture of proteins extracted from normal mouse liver cytoplasm sample via integration with 2D-LC-ESI-MS/MS to show its potential application for large-scale protein identification.     

Functionalized magnetic micro- and nanoparticles: optimization and application to micro-chip tryptic digestion

The preparation of an easily replaceable protease microreactor for micro-chip application is described. Magnetic particles coated with poly(N-isopropylacrylamide), polystyrene, poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate), poly(glycidyl methacrylate), [(2-amino-ethyl)hydroxymethylen]biphosphonic acid, or alginic acid with immobilized trypsin were utilized for heterogeneous digestion. The properties were optimized, with the constraint of allowing immobilization in a microchannel by a magnetic field gradient. To obtain the highest digestion efficiency, sub-micrometer spheres were organized by an inhomogeneous external magnetic field perpendicularly to the direction of the channel. Kinetic parameters of the enzyme reactor immobilized in micro-chip capillary (micro-chip immobilized magnetic enzyme reactor (IMER)) were determined. The capability of the proteolytic reactor was demonstrated by five model (glyco)proteins ranging in molecular mass from 4.3 to 150 kDa. Digestion efficiency of proteins in various conformations was investigated using SDS-PAGE, HPCE, RP-HPLC, and MS. The compatibility of the micro-chip IMER system with total and limited proteolysis of high-molecular-weight (glyco)proteins was confirmed. It opens the route to automated, high-throughput proteomic micro-chip devices.     

On-column tryptic mapping of proteins using metal-ion-chelated magnetic silica microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Peptide mapping analysis, utilizing an easily replaceable and regenerable on-column enzymatic microreactor with metal-ion-chelated adsorption of enzyme on magnetic silica microspheres, combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), was developed. Firstly, magnetic microspheres of small size and strong magnetism were prepared through solvothermal reaction. Thereafter, by introducing tetraethyl orthosilicate (TEOS), magnetic silica (MS) microspheres were formed. Trypsin could then be immobilized onto the MS microspheres based on the Lewis acid-base interaction through the divalent cation chelators such as iminodiacetic acid (IDA), which was chemically bound to the microspheres through the introduction of glycidoxypropyltrimethoxysilane (GLYMO). The trypsin-immobilized MS microspheres were then locally packed into the capillary by the application of a strong magnetic field using a magnet. The performance of the method was exemplified with digestion of bovine serum albumin for 5 min at 50 degrees C and the result was comparable to the 12 h in-solution digestion. The ability of regeneration of the prepared on-column microreactor and good reproducibility of microreactor before and after regeneration were also demonstrated.     

膜亲和色谱用于胰蛋白酶抑制剂的分离

分别采用酰化-胺化和氯甲基化-胺化对聚砜进行化学修饰,用相转化法制成平均孔径为450nm的超滤膜,经重氮化反应,共价键合上具有活性的胰蛋白酶。其相对活力可达10200 u/g。每克化学改性聚砜膜上可固载化上15 mg胰蛋白酶。用此酶膜对胰蛋白酶抑制剂进行了亲和分离,一次可得6.5mg纯胰蛋白酶抑制剂。     

Off-line form of the Michaelis-Menten equation for studying the reaction kinetics in a polymer microchip integrated with enzyme microreactor

We firstly transformed the traditional Michaelis-Menten equation into an off-line form which can be used for evaluating the Michaelis-Menten constant after the enzymatic reaction. For experimental estimation of the kinetics of enzymatic reactions, we have developed a facile and effective method by integrating an enzyme microreactor into direct-printing polymer microchips. Strong nonspecific adsorption of proteins was utilized to effectively immobilize enzymes onto the microchannel wall, forming the integrated on-column enzyme microreactor in a microchip. The properties of the integrated enzyme microreactor were evaluated by using the enzymatic reaction of glucose oxidase (GOx) with its substrate glucose as a model system. The reaction product, hydrogen peroxide, was electrochemically (EC) analyzed using a Pt microelectrode. The data for enzyme kinetics using our off-line form of the Michaelis-Menten equation was obtained (K(m) = 2.64 mM), which is much smaller than that reported in solution (K(m) = 6.0 mM). Due to the hydrophobic property and the native mesoscopic structure of the poly(ethylene terephthalate) film, the immobilized enzyme in the microreactor shows good stability and bioactivity under the flowing conditions.     

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.     

Membrane affinity chromatography used for the separation of trypsin inhibitor.

Polysulphone (PS) was chemically modified by acrylation-amination and by chloromethylation-amination, respectively. An ultrafiltration membrane of chemically modified polysulphone (CMPS) was prepared by the phase inversion method. Trypsin was then covalently bonded onto the CMPS membrane by diazotization. The activity of immobilized trypsin reaches up to 10200 U/g; 15 mg trypsin was immobilized on 1 g CMPS membrane. Separation of soybean trypsin inhibitor was carried out on the affinity membrane, yielding 6.5 mg pure trypsin inhibitor in one run. The enzyme membrane has good activity and stability.     

Zeolite nanoparticle modified microchip reactor for efficient protein digestion

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol-gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon-oxygen-silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s(-1). Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 microL min(-1), which affords a very short reaction time of less than 5 s.