Optimization of a nano‐enzymatic reactor for on‐line tryptic digestion of polypeptide conjugates by capillary electrophoresis

This work aims at studying the optimization of an on‐line capillary electrophoresis (CE)‐based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on‐line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.     

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.     

Triamine‐bonded stationary phase for open tubular capillary electrochromatography

A multi‐functional separation column modified with 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane was developed for open tubular capillary electrochromatography. This functional hydrophilic triamine‐bonded open tubular column could generate both anodic and cathodic EOF. When the pH of the running buffer was below 5.3 (30% 3‐[2‐(2‐aminoethylamino)ethylamino] propyl‐trimethoxysilane, v/v), the anodic EOF was exhibited, which greatly prevented the undesired adsorptions of basic proteins on the capillary inner wall. Favorable separation of four basic proteins (viz. trypsin, ribonuclease A, lysozyme and cytochrome c) was successfully achieved at pH 3.5 of 10 mmol/L phosphate buffer. The column efficiencies of proteins were in the range from 87 000 to 110 000 plates/m, and the RSD values for migration time of four proteins were less than 1.2% (run‐to‐run, n=5). The ionic analytes were also separated efficiently in the co‐electroosmotic mode. The average efficiencies ranged from 81 000 to 190 000 plates/m for seven aromatic acids and 186 000–245 000 plates/m for four nucleoside monophosphates, respectively, and good capillary column repeatability was gained with RSD of the migration time not more than 3.0%. The triamine‐bonded open tubular capillary column is favorable to be an alternative functional medium for the further analysis of basic proteins and anionic analytes.     

In-line liquid-phase microextraction for selective enrichment and direct electrophoretic analysis of acidic drugs

This work describes an efficient in-line extraction-preconcentration unit coupled to the electrophoretic capillary based on a liquid-phase microextraction (LPME) process, which can be directly assembled to the cartridge of the commercial CE equipment. The unit permits analyte extraction, preconcentration and electrophoretic separation to be automatically performed in the commercial CE equipment without the need for additional hardware or software. This new approach was usefully used for the separation and determination of nonsteroidal anti-inflammatory drugs in human urine permitting at least to analyze 30 consecutive real samples. The LODs were lower than 2 microg/L and the reproducibility, expressed as RSD, was 3.1% for the same unit and only 4.8% between different units.     

In‐capillary microextraction using monolithic polymers: Application to preconcentration of carbamate pesticides prior to their separation by MEKC

In the present work we report a novel procedure for in‐capillary microextraction using a monolithic polymeric sorbent. In the proposed methodology, sample treatment takes place in the CE instrument but in a different capillary from that used for the electrophoretic separation. Polymers based on butyl methacrylate and divinylbenzene formed in situ inside a capillary column were assayed. The best results were found with the divinylbenzene‐based polymers. The usefulness of the proposed procedure was checked for the determination of carbamate pesticides (Methomyl, Asulam, Carbendazim, Aldicarb, Carbetamide, Propoxur, Pirimicarb, Carbaryl, Carbofuran and Methiocarb) and three of their degradation compounds (Aldicarb sulphoxide, 2‐isopropoxyphenol and α‐naphthol) using MEKC. The optimization of the MEKC is reported, a good separation of the 13 analytes being obtained in less than 6 min. The analytical method using in‐capillary microextraction was validated in terms of linearity, repeatability, precision (RSD≤18% for 50 μg/L), and LODs (1–16 μg/L), and it revealed the usefulness of this in‐capillary preconcentration procedure for the determination of analytes of intermediate polarity.     

Coupling of biological sample handling and capillary electrophoresis.

The analysis of biological samples (e.g., blood, urine, saliva, tissue homogenates) by capillary electrophoresis (CE) requires efficient sample preparation (i.e., concentration and clean-up) procedures to remove interfering solutes (endogenous/exogenous and/or low-/high-molecular-mass), (in)organic salts and particulate matter. The sample preparation modules can be coupled with CE either off-line (manual), at-line (robotic interface), on-line (coupling via a transfer line) or in-line (complete integration between sample preparation and separation system). Sample preparation systems reported in the literature are based on chromatographic, electrophoretic or membrane-based procedures. The combination of automated sample preparation and CE is especially useful if complex samples have to be analyzed and helps to improve both selectivity and sensitivity. In this review, the different modes of solid-phase (micro-) extraction will be discussed and an overview of the potential of chromatographic, electrophoretic (e.g., isotachophoresis, sample stacking) and membrane-based procedures will be given.     

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.     

Stable physically adsorbed coating of poly-vinyl alcohol for the separation of basic proteins

In aqueous capillary electrophoresis, the electroosmotic flow can be strongly suppressed by coating the inner surface of the capillary. In the present work hydrophilic coating of 4% polyvinyl alcohol (PVA) has been used for the analysis of basic proteins. The coating is simple and easy to obtain. The separation of ribonuclease and α-chymotrypsin has been uniquely done with other three basic proteins (lysozyme, cytochrome-c and trypsin) using a buffer 11.60 mM sodium acetate and 18.40 mM acetic acid at pH 4.5 in addition to positive power supply of 20 kV at 25°C. Detection was performed using UV detector at 230 nm. The proposed PVA coated capillary provides reproducible separation of five basic proteins within 10 min with RSD values for mobility bellow 1.4% (n = 6) for all the five basic proteins. The stability of coated capillary has been checked up to 40 runs. The viscosity measurement for 4% PVA have been studied and scanning electron microscope (SEM) images obtained to make it compatible with future micro-chip applications.     

A new sequential injection analysis‐capillary electrophoresis system with amperometric detection

A novel and fully automated sequential injection analysis manifold coupled to a capillary electrophoresis apparatus with amperometric detection, is described. The sequential injection manifold was isolated from the high voltage by inserting an air plug into the circuit. Small buffer reservoirs were used to avoid the need to pump fresh buffer to the interface during the electrophoretic separation. No decoupling device was used to mitigate the interference from the high voltage electric field, instead the potential shift induced by the separation voltage, was accounted for. The new hydrodynamic injection method presented is based on the overpressure created in the circuit when a pinch valve is closed for a predetermined time. The injection method yields RSD values of peak height and area below 2.55 and 1.82%, respectively, at different durations of valve closure (n = 5). The capillary and working electrode alignment was achieved by adapting a commercial available capillary union. When the electrode was replaced, the alignment method proved to be very reliable, yielding RSD values of peak height and area lower than 2.64 and 2.08%, respectively (n = 8). Using this system with a gold microelectrode, dopamine, and epinephrine could be quantified within the concentration range of 1–500 μM and detected at a concentration of 0.3 μM. The methods here presented could be applied for the development of new capillary electrophoresis systems with amperometric detection and/or to the design of fully automated systems for online process monitoring purposes.     

Improving the sensitivity of the determination of ceftiofur by capillary electrophoresis in environmental water samples: in-line solid phase extraction and sample stacking techniques

This paper describes two different approaches for increasing the sensitivity for the analysis of ceftiofur by capillary electrophoresis (CE). Two different techniques based on the introduction of an enlarged volume of sample, namely large volume sample stacking (LVSS) and in-line solid phase extraction (SPE) were studied and compared. LVSS allowed the on-column electrophoretic preconcentration of ceftiofur without modification of the separation capillary. In-line SPE-CE was developed by using a home-made microcartridge that was filled with a reversed-phase sorbent (C₁₈). The microcartridge was coupled in-line near the inlet of the separation capillary. LVSS and in-line SPE-CE allowed automated operation and improved sensitivity for the analysis of ceftiofur with respect to conventional CE. When environmental water samples were analyzed, an additional pretreatment step based on off-line SPE was necessary in both cases to further decrease the detection limits. In terms of sensitivity for the determination of ceftiofur in river water samples, the combination of off-line SPE with in-line SPE-CE was found the most sensitive with a detection limit of 10 ng L⁻¹, whereas the method based on the use of off-line SPE with LVSS presented a detection limit of 100 ng L⁻¹.     

基于电驱动在线快速分离富集技术的研究进展

样品前处理能将待测物从复杂基质中预先分离富集出来,以提高分析方法的灵敏度、选择性和准确性,是复杂样品分析的关键步骤。样品前处理是一个非自发的、从无序到有序的熵减过程,不仅费时费力,还极易引起误差。向体系输入能量和降低体系熵值可以增强分离富集效果,加快样品制备过程。将电场引入在线样品前处理,既能向体系做功,又能驱动样品定向迁移,使前处理的熵减过程快速顺利进行,是快速样品制备的有效途径。基于电驱动的在线分离富集技术综合了多种加速策略：（1）以电场形式向体系输入能量,加速传质和传热过程;（2）采用电渗流、电泳等电驱动定向流实现样品在分离、富集、检测各步骤之间的定向迁移,保证样品前处理与检测顺利进行;（3）利用在线联用技术集成样品前处理与分析检测各步骤,从而提高自动化程度,减少人为误差;（4）通过微型化装置或微萃取方法提高样品制备效率,缩短样品制备时间。该文总结了近10年与基于电驱动的在线快速分离富集技术相关的90多篇文献,综述了该技术领域的研究进展,探讨了电驱动毛细管在线快速分离富集技术、电驱动芯片在线快速分离富集技术和电驱动膜萃取在线分离富集技术各自的优势和潜力,并展望了该类技术的发展与应用趋势。     

赖氨酸C端内切酶/胰蛋白酶顺序酶切在蛋白质组学样本制备中的评估

采用胰蛋白酶(Trypsin)单独酶切与不同酶量的赖氨酸C端内切酶(Lys-C/trypsin)顺序酶切两种方法,对293T细胞全蛋白样本进行酶解消化,系统评估Lys-C/trypsin顺序酶切与Trypsin单一酶切在蛋白质组学样本制备中的差别.实验结果表明,Lys-C/trypsin顺序酶切不仅能显著提高肽段和蛋白质的鉴定数目,同时降低遗漏K酶切位点的数目及比例,而且得到的肽段长度有利于质谱鉴定,蛋白质覆盖率明显提升.通过对酶的用量进行优化对比,最终确定了Lys-C/trypsin顺序酶切时酶的合理用量.本研究结果对提高蛋白质组学样本的制备质量以及蛋白质的序列鉴定覆盖度具有指导意义.     

Effects of common surfactants on protein digestion and matrix-assisted laser desorption/ionization mass spectrometric analysis of the digested peptides using two-layer sample preparation

While surfactants are commonly used in preparing protein samples, their presence in a protein sample can potentially affect the enzymatic digestion process and the subsequent analysis of the resulting peptides by mass spectrometry. The extent of the tolerance of matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) to surfactant interference in peptide analysis is very much dependent on the matrix/sample preparation method. In this work the effects of four commonly used surfactants, namely n-octyl glucoside (OG), Triton X-100 (TX-100), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and sodium dodecyl sulfate (SDS), for biological sample preparation on trypsin digestion and MALDI-MS of the resulting digest are examined in detail within the context of using a two-layer method for MALDI matrix/sample preparation. Non-ionic and mild surfactants, such as OG, TX-100 or CHAPS, are found to have no significant effect on trypsin digestion with surfactant concentrations up to 1%. However, TX-100 and CHAPS interfere with the subsequent peptide analysis by MALDI-MS and should be removed prior to peptide analysis. OG is an MS-friendly surfactant and no effect is observed for MALDI peptide analysis. The effect of SDS on trypsin digestion in terms of the number of peptides generated and the overall protein sequence coverage by these peptides is found to be protein dependent. The use of SDS to solubilize hydrophobic membrane proteins, followed by trypsin digestion in the presence of 0.1% SDS, results in a peptide mixture that can be analyzed directly by MALDI-MS. These peptides are shown to provide better sequence coverage compared with those obtained without the use of SDS in the case of bacteriorhodopsin, a very hydrophobic transmembrane protein. This work illustrates that MALDI-MS with the two-layer sample preparation method can be used for direct analysis of protein digests with no or minimum sample cleanup after proteins are digested in a solution containing surfactants.     

Capillary Electrophoretic Separation of Tricyclic Antidepressants Using 1‐Alkyl‐3‐Methylimidazolium‐Based Ionic Liquids as Background Electrolyte

A capillary electrophoretic (CE) method coupled with the use of 1‐ethyl‐3‐methylimidazolium tetrafluoroborate (1E‐3MI‐TFB) ionic liquid as background electrolyte (BGE) has been developed for the simultaneous separation of nine tricyclic antidepressants, viz. amitriptyline (Ami), clomipramine (Clo), desipramine (Des), fluphenazine (Flu), imipramine (Imi), nortriptyline (Nor), promazine (Pro), thioridazine (Thi) and trimipramine (Tri). Resolution of TCAs with similar molecular structures and pK_a values was accomplished by minute manipulation of the electrophoretic velocities of TCAs via reversed electroosmotic flow (EOF) generated by adsorption of 1E‐3MI cations onto the capillary wall. The optimal separation was obtained with a 50 mM 1E‐3MI‐TFB as the sole BGE at pH 3. Symmetric peaks with efficiencies up to 2.4 × 10⁵ plates/m were achieved. RSD values on migration times and peak areas were in the ranges of 0.63–0.95% and 3.41–6.34% (n = 4), respectively. The role of different alkyl groups on the imidazolium cations was also investigated.     

Development of an open-tubular trypsin reactor for on-line digestion of proteins

A study was initiated to construct a micro-reactor for protein digestion based on trypsin-coated fused-silica capillaries. Initially, surface plasmon resonance was used both for optimization of the surface chemistry applied in the preparation and for monitoring the amount of enzyme that was immobilized. The highest amount of trypsin was immobilized on dextran-coated SPR surfaces which allowed the covalent coupling of 11 ng mm⁻² trypsin. Fused-silica capillaries were modified in a similar manner and the resulting open-tubular trypsin-reactors having a pH optimum of pH 8.5, display a high activity when operated at 37 °C and are stable for at least two weeks when used continuously. Trypsin auto-digestion fragments, sample carry-over, and loss of signal due to adsorption of the protein were not observed. On-line digestion without prior protein denaturation, followed by micro-LC separation and photodiode array detection, was tested with horse-heart cytochrome C and horse skeletal-muscle myoglobin. The complete digestion of 20 pmol μL⁻¹ horse cytochrome C was observed when the average residence time of the protein sample in a 140 cm ×50 μm capillary immobilized enzyme reactor (IMER) was 165 s. Mass spectrometric identification of the injected protein on the basis of the tryptic peptides proved possible. Protein digestion was favorable with respect to reaction time and fragments formed when compared with other on-line and off-line procedures. These results and the easy preparation of this micro-reactor provide possibilities for miniaturized enzyme-reactors for on-line peptide mapping and inhibitor screening.     

Pepsin‐modified chiral monolithic column for affinity capillary electrochromatography

Pepsin‐modified affinity monolithic capillary electrochromatography, a novel microanalysis system, was developed by the covalent bonding of pepsin on silica monolith. The column was successfully applied in the chiral separation of (±)‐nefopam. Furthermore, the electrochromatographic performance of the pepsin‐functionalized monolith for enantiomeric analysis was evaluated in terms of protein content, pH of running buffer, sample volume, buffer concentration, applied voltage, and capillary temperature. The relative standard deviation (%RSD) values of retention time (intraday <0.53, n = 10; interday <0.53, n = 10; column‐to‐column <0.70, n = 20; and batch‐to‐batch <0.80, n = 20) indicated satisfactory stability of these columns. No appreciable change was observed in retention and resolution for chiral recognition of (±)‐nefopam in 50 days with 100 injections. The proteolytic activity of this stationary phase was further characterized with bovine serum albumin as substrate for online protein digestion. As for monolithic immobilized enzyme reactor, successive protein injections confirmed both the operational stability and ability to reuse the bioreactor for at least 20 digestions. It implied that the affinity monolith used in this research opens a new path of exploring particularly versatile class of enzymes to develop enzyme‐modified affinity capillary monolith for enantioseparation.     

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.     

High-Throughput Proteomics Enabled by a Photocleavable Surfactant

Mass spectrometry (MS)-based proteomics provides unprecedented opportunities for understanding the structure and function of proteins in complex biological systems; however, protein solubility and sample preparation before MS remain a bottleneck preventing high-throughput proteomics. Herein, we report a high-throughput bottom-up proteomic method enabled by a newly developed MS-compatible photocleavable surfactant, 4-hexylphenylazosulfonate (Azo) that facilitates robust protein extraction, rapid enzymatic digestion (30 min compared to overnight), and subsequent MS-analysis following UV degradation. Moreover, we developed an Azo-aided bottom-up method for analysis of integral membrane proteins, which are key drug targets and are generally underrepresented in global proteomic studies. Furthermore, we demonstrated the ability of Azo to serve as an “all-in-one” MS-compatible surfactant for both top-down and bottom-up proteomics, with streamlined workflows for high-throughput proteomics amenable to clinical applications.     

Surfactant‐based ionic liquids for extraction of phenolic compounds combined with rapid quantification using capillary electrophoresis

A rapid liquid phase extraction employing a novel hydrophobic surfactant‐based room temperature ionic liquid (RTIL), tetrabutylphosphonium dioctyl sulfosuccinate ([4C₄P][AOT]), coupled with capillary electrophoretic‐UV (CE‐UV) detection is developed for removal and determination of phenolic compounds. The long‐carbon‐chain RTIL used is sparingly soluble in most solvents and can be used to replace volatile organic solvents. This fact, in combination with functional‐surfactant‐anions, is proposed to reduce the interfacial energy of the two immiscible liquid phases, resulting in highly efficient extraction of analytes. Several parameters that influence the extraction efficiencies, such as extraction time, RTIL type, pH value, and ionic strength of aqueous solutions, were investigated. It was found that, under acidic conditions, most of the investigated phenols were extracted from aqueous solution into the RTIL phase within 12 min. Good linearity was observed over the concentration range of 0.1–80.0 μg/mL for all phenols investigated. The precision of this method, expressed as RSD, was determined to be within 3.4–5.3% range. The LODs (S/N = 3) of the method were in the range of 0.047–0.257 μg/mL. The proposed methodology was successfully applied to determination of phenols in real water samples.     

Multisyringe flow injection analysis coupled to capillary electrophoresis (MSFIA-CE) as a novel analytical tool applied to the pre-concentration, separation and determination of nitrophenols

For the first time, a multisyringe flow injection analysis capillary electrophoresis system is described. The potential of the hyphenation for sample treatment including analyte pre-concentration is demonstrated by its successful application to the determination of mono-nitrophenols (NPs) in different water samples. The analytical system was used to automate in-line sample acidification, analyte pre-concentration, elution, hydrodynamic injection, electrophoretic separation, and detection as well as the maintenance and re-conditioning of the solid-phase extraction (SPE) column and the separation capillary. A pre-concentration factor of better than 115 and detection down to 0.11 micromol L(-1) were achieved. Detection was carried out at visible wavelength using a blue LED as a low baseline-noise light source. High repeatability was obtained each for migration times and for peak heights with relative standard deviations typically below 2.5 and 6% including the pre-concentration procedure, respectively. Three injections per hour were achieved by running in parallel the pre-concentrating and the electrophoretic separation procedures. Instrumental control and data registration and evaluation were carried out with the software package AutoAnalysis, allowing autonomous operation of the analytical system.