Capillary Electrophoresis Mode Research Articles

Capillary electrophoresis-mass spectrometry for intact protein analysis: Pharmaceutical and biomedical applications (2018-March 2023).

Capillary electrophoresis is recognized as a valued separation technique for its high separation efficiency, low sample consumption, good economic and ecological aspects, reproducibility, and complementarity to traditional liquid chromatography techniques. Capillary electrophoresis experiments are generally performed utilizing optical detection, such as ultraviolet or fluorescence detectors. However, in order to provide structural information, capillary electrophoresis hyphenated to highly sensitive and selective mass spectrometry has been developed to overcome the limitations of optical detections. Capillary electrophoresis-mass spectrometry is increasingly popular in protein analysis, including biopharmaceutical and biomedical research. It is frequently applied for the determination of physicochemical and biochemical parameters of proteins, offers excellent performance for in-depth characterizations of biopharmaceuticals at various levels of analysis, and has been also already proven as a promising tool in biomarker discovery. In this review, we focus on the possibilities and limitations of capillary electrophoresis-mass spectrometry for protein analysis at their intact level. Various capillary electrophoresis modes and capillary electrophoresis-mass spectrometry interfaces, as well as approaches to prevent protein adsorption and to enhance sample loading capacity, are discussed and the recent (2018-March 2023) developments and applications in the field of biopharmaceutical and biomedical analysis are summarized.

Advances in the Applications of Capillary Electrophoresis to Tobacco Analysis

Background: Capillary electrophoresis (CE) has found a wide range of applications because of its high separation efficiency, low expense, short analysis time and minimal sample volume requirement. The tobacco quality depends on the nature and quantity of numerous substances. CE has been applied in the constituent analysis of tobacco and tobacco products for quality control and tobacco research. Methods: The advances in the applications of CE to tobacco analysis are reviewed. The main subjects cover the separation modes of CE, the detection techniques of CE, sample preparations and the applications of CE in the measurements of various constituents in tobacco samples. In addition, the CE-based metabonomic investigation of tobacco is also introduced. Results: Capillary zone electrophoresis, micellar electrokinetic chromatography, capillary isotachophoresis, capillary gel electrophoresis, capillary electrochromatography and non-aqueous CE have been applied in the determination of a variety of constituents in tobacco and tobacco products. The assayed substances include alkaloids, amines, saccharides, organic acids, inorganic ions, phenols, phenolic acids, flavonoids, amino acids, peptides, proteins, hormones, agricultural chemicals, etc. Conclusion: This review demonstrates that CE is a promising analytical technique in the field of tobacco analysis. It is anticipated that CE will find more and more applications in tobacco investigations.

Stronger together: Analytical techniques for recombinant adeno associated virus

With recent FDA approval of two recombinant adeno‐associated virus (rAAV)‐based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well‐established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

Rapid Screening and Estimation of Binding Constants for Interactions of Fe3+ with Two Metalloproteins, Apotransferrin and Transferrin, Using Affinity Mode of Capillary Electrophoresis

The interaction behavior of Fe3+ with transferrin and apotransferrin (iron-free form) was investigated in this study using affinity capillary electrophoresis. Change in the mass and charge of protein upon binding to the metal ion in the capillary tube led to variation in its migration time and was used to measure the noncovalent binding interactions by fast screening method. Acetanilide was used as the electroosmotic flow (EOF) marker to avoid possible errors due to the change in EOF during the experiment. The binding results were calculated from the mobility ratios of protein (Ri) and EOF marker (Rf) using the formula (Ri − Rf)/Rf or ∆R/Rf. For more comprehensive understanding, the kinetics of the interaction was studied and binding constants were calculated. Results showed that the Fe3+ displayed insignificant interaction with both proteins at lower metal ion concentrations (5–25 μmol/mL). However, transferrin exhibited significant interactions with the metal ion at 50 and 100 μmol/mL (ΔR/Rf = 0.0114 and 0.0201, resp.) concentrations and apotransferrin showed strong binding interactions (ΔR/Rf = −0.0254 and 0.0205, resp.) at relatively higher Fe3+ concentrations of 100 and 250 μmol/mL. The binding constants of 18.968 mmol−1 and −13.603 mmol−1 were recorded for Fe3+ interaction with transferrin and apotransferrin, respectively, showing significant interactions. Different binding patterns of Fe3+ with both proteins might be attributed to the fact that the iron-binding sites in transferrin have already been occupied, which was not the case in apotransferrin. The present study may be used as a reference for the investigation of protein-metal ion, drug-protein, drug-metal ion, and enzyme-metal ion interactions and may be helpful to provide preliminary insight into the new metal-based drug development.

Pseudophase microextraction for in-line sample concentration in micellar electrokinetic chromatography

Pseudophase microextraction (PPME) as a simple in-line sample concentration technique in micellar electrokinetic chromatography (MEKC) is presented. In contrast to popular electric-field driven stacking techniques in MEKC such as sweeping, PPME is pressure-driven. The technique afforded up to 403-2968x improvements in peak heights for fenoprop, dichlorprop, 1- and 2-naphthol compared to typical injection. Under the same MEKC conditions, the improvements in PPME were up to 23-59x better compared to sweeping. Briefly in PPME, the entire capillary was loaded (up to 20 capillary volumes) with the sample prepared in a dilute solution of cetyltrimethylammonium bromide ([CTAB] > critical surface aggregation concentration). The CTAB formed aggregates at the inner capillary walls and these aggregates acted as a stationary chromatographic pseudophase. After clean-up via flushing the capillary with purified water, the MEKC background solution (BGS) with sodium dodecyl sulfate was then introduced by pressure from the outlet end to elute the retained analytes. The analytes concentrate at front of the BGS and the front was moved to the inlet end of the capillary prior to MEKC. Optimization strategies and current limitations in PPME-MEKC are described. The linear ranges using a 4 capillary volume sample load obtained for fenoprop, dichlorprop, 1- and 2-naphthol were between 1 and 160 ng/mL (r2s ≥ 0.996), LOQs = 1–2.5 ng/mL and repeatability %RSDs (n = 6) were ≤5% (intra-day) and ≤7% (inter-day) (using low analyte concentrations 1-5x LOQ). PPME-MEKC with simple dilution of fortified real samples (no off-line sample concentration) was also able to detect low levels of dichlorprop (10 ng/mL, limit set in Australia) and 1- and 2−naphthol (7.5–15 ng/mL) in a drinking water and natural water sample, respectively (% recovery = 84–108%). The concept of PPME may find use in other modes of capillary electrophoresis and other nano-microscale separations.

Recent biopharmaceutical applications of capillary electrophoresis methods on recombinant DNA technology-based products.

Biopharmaceuticals (recombinant technology-based products, vaccines, whole blood and blood components, gene therapy, cells, tissues, etc.,) are described as biological medical products produced from various living sources such as human, microbial, animal, and so on by manufacturing, extraction, or semi-synthesis. They are complex molecules having high molecular weights. For their safety and efficacy, their structural, clinical, physicochemical, and chemical features must be carefully controlled, and they must be well characterized by analytical techniques before the approval of the final product. Capillary electrophoresis (CE) having versatile modes can provide valuable safety and efficacy information, such as amino acid sequence, size variants (low and high molecular weight variants), charged variants (acidic and basic impurities), aggregates, N-linked glycosylation, and O-linked glycosylation. There are numerous applications of CE in the literature. In this review, the most significant and recent studies on the analysis of recombinant DNA technology-based products using different CE modes in the last ten years have been overviewed. It was seen that the researches mostly focus on the analysis of mAbs and IgG. In addition, in recent years, researchers have started to prefer CE combined mass spectrometry (MS) techniques to provide a more detailed characterization for protein and peptide fragments.

Annual review of capillary electrophoresis technology in 2020

该文为2020年毛细管电泳(capillary electrophoresis, CE)技术年度回顾。归纳总结了以“capillary electrophoresis-mass spectrometry”或“capillary isoelectric focusing”或“micellar electrokinetic chromatography”或“capillary electrophoresis”为关键词在ISI Web of Science数据库中进行主题检索得到的2020年CE技术相关研究论文222篇,以及中文期刊《分析化学》和《色谱》中CE技术相关的研究论文37篇。对2020年影响因子(IF)≥5.0的Analytical Chemistry, Food Chemistry, Analytica Chimica Acta和Talanta等13本期刊的38篇文章报道的科研工作作了逐一介绍;对IF<5.0的期刊中CE技术报道较为集中的Journal of Chromatography A和Electrophoresis两本分析化学类期刊发表40篇文章中的代表性内容作了综合介绍;对重要的中文期刊《分析化学》出版的“核酸适配体专刊”和《色谱》出版的2期CE技术专刊所收录的37篇文章中的工作作了总体介绍。总体来说,2020年CE技术发展趋势仍以毛细管电泳-质谱(CE-MS)的新方法和新应用最为突出,主要集中在CE-MS与电化学检测、固相萃取以及多种毛细管电泳模式的联用方面,CE-MS接口相关的报道较前几年有所减少;常规CE技术则以胶束电动毛细管色谱(MEKC)在复杂样本分析、浓缩富集应用为主,尤其在食品和药品等复杂基质样本分析方面的报道较为集中;此外,我国CE相关领域专家学者的科研成果涵盖了CE在生命科学、临床医学、医药研发、环境科学、天然产物、食品分析等领域的应用,代表了国内CE科研应用水平和现状。

Protein mapping of peanut extract with capillary electrophoresis.

Protein separation can be achieved with different modes of capillary electrophoresis, such as with capillary gel electroporesis (CGE) or with capillary zone electrophoresis (CZE). CZE protein mapping of peanut extract was approached in four different ways, combining neutral-coated or multilayer-coated capillaries with pHs well over or under the isoelectric point range of the proteins of interest. At acidic pHs, the mobility ranges of the major peanut allergens Ara h1, Ara h2, Ara h3, and Ara h6 were identified. Although the pH is a major factor in CZE separation, buffers with different compositions but with the same pH and ionic strength showed significantly different resolutions. Different components of the electrolyte were studied in a multifactorial design of experiment. CE-SDS and CZE proved to be suitable for protein mapping and we were able to distinguish different batches of peanut extract and burned peanut extract.

Pre-Concentration and Analysis of Mycotoxins in Food Samples by Capillary Electrophoresis.

Mycotoxins are considered one of the most dangerous agricultural and food contaminants. They are toxic and the development of rapid and sensitive analytical methods to detect and quantify them is a very important issue in the context of food safety and animal/human health. The need to detect mycotoxins at trace levels and to simultaneously analyze many different mycotoxin types became mandatory to protect public health. In fact, European Commission regulations specified both their limits in foodstuffs and official sample preparation protocols in addition to analytical methods to verify their presence. Capillary Electrophoresis (CE) includes different separation modes, allowing many versatile applications in food analysis and safety. In the context of mycotoxins, recent advances to improve CE sensitivity, particularly pre-concentration techniques or miniaturized systems, deserve remarkable attention, as they provide an interesting approach in the analysis of such contaminants in complex food matrices. This review summarizes the applications of CE combined with different pre-concentration approaches, which have been proposed in the literature (mainly) in the last ten years. A section is also dedicated to recent microchip–CE devices since they represent the most promising CE mode for this application.

Advances in Lipid Capillary Electromigration Methods to Food Analysis Within the 2010s Decade

Capillary electrophoresis (CE) is a technique that may offer high-resolution separations in complex matrices with a short analysis time. It generally employs straightforward sample preparation protocols and requires low amounts of sample and reagents, which follows the green chemistry concept. This review presents several methods developed within the 2010s decade, aiming simpler and faster food analyses in the determination of lipids applying CE. It summarizes sample preparation protocols, background electrolyte, and instrumental conditions optimized in each method. Different CE modes and detection systems are discussed, as well as figures of merit of the developed analytical methods. Moreover, it approaches significant challenges involved with lipid assay by CE and opportunities for innovation in the lipid research.

Extraction and preconcentration of compounds from the l-tyrosine metabolic pathway prior to their micellar electrokinetic chromatography separation

The prominent biological effects of adrenaline (A), noradrenaline (NA) and dopamine (DA) as well as the clinical importance of their metabolites (such as dihydroxyphenylacetic acid (DOPAC), methoxy‑4-hydroxyphenyl glycol (MHPG), dihydroxyphenylglycol (DHPG), metanephrine (M), normetanephrine (NM), vanillylmandelic acid (VMA), homovanillic acid (HVA)) have forced researchers to evaluate new analytical methodologies for their isolation and preconcentration from biological samples. For this reason, the three most popular extraction techniques (dispersive liquid-liquid microextraction (DLLME), solid-phase extraction (SPE), solid-phase microextraction (SPME)) were tested. Micellar electrokinetic chromatography (MEKC) – a mode of capillary electrophoresis – with a diode array detector (DAD) was applied to assess the extraction efficiency. Next, the enrichment factor (EF) of each applied method was calculated in respect to standard mixtures of the analytes at the same concentration levels. The EF results of seven selected metabolites of biogenic amines (BAs) from urine after sample preparation procedures based on twenty-five different protocols (one DLLME, thirteen SPE and eleven SPME) were calculated and compared using hierarchical cluster analysis (HCA). The SPE as well as SPME procedures were proved to be the most effective approaches for the simultaneous extraction of the chosen compounds. Moreover, an ionic liquid (IL) – 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide – added to methanol in SPME additionally could successfully improve the extraction efficiency. It was also confirmed that the HCA approach could be considered a supportive tool in the selection of a suitable sample preparation procedure for that group of endogenous substances.

Study of interactions between carboxylated core shell magnetic nanoparticles and polymyxin B by capillary electrophoresis with inductively coupled plasma mass spectrometry

In this work, interactions of carboxylated core shell magnetic nanoparticles with polymyxin B sulfate were studied by connecting capillary electrophoresis with inductively coupled plasma mass spectrometry. The interaction was probed by affinity mode of capillary electrophoresis with 25 mM phosphate buffer at physiological pH. 54Fe, 56Fe, 57Fe, 34S, and 12C isotopes were used to monitor the migration of an electroosmotic flow marker and the interaction of the nanoparticles with polymyxin B. The analysis of interaction data showed two distinct interaction regions, one with low polymyxin B concentration, the second with high polymyxin B concentration. These regions differed in the strength of the interaction, 1.49 × 107 M−1 and 1.60 × 104 M−1, and in the stoichiometry of 0.7 and 3.5, respectively. These differences can be explained by the decrease of electrostatic repulsion between nanoparticles caused by polymyxin B. This is also in agreement with the nanoparticles peak shapes: sharp for low polymyxin B concentrations and broad for high polymyxin B concentrations.

High separation efficiency of gold nanomaterials of different aspect ratio and size using capillary transient isotachophoresis

Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP), were compared for the detection and separation of spherical gold nanoparticles (AuNPs) and gold nanorods (AuNRs). The development of ctITP using two different leading ions is described. Overall, when compared to traditional capillary zone electrophoresis (CZE), ctITP resulted in improved peak shape and peak efficiency. Specifically, the number of theoretical plates for AuNR samples increased by a factor of 2–2.5 depending on the choice of leading ion. Further, using ctITP two AuNRs differing by aspect ratio were baseline resolved, whereas the same AuNRs could not be separated using CZE or other techniques like single particle inductively coupled plasma mass spectrometry (spICP-MS) and asymmetric flow field-flow fractionation (AF4). The results of this study demonstrate that ctITP is an efficient on-line technique for the improved detection and separation of gold nanomaterials in CE.

Poly-4-vinylpiridinium nanosponges as modifiers of the electrophoretic systems for the charged analytes separation

Novel hydrophilic ionic polymer nanoparticles based on the N-alkylated hypercrosslinked poly-4-vinylpyridine (nanosponges ‒ NS ) have porous structure, pH-independent positive charge and contain the aromatic rings. These nanosponges have not yet been studied in the capillary electrophoresis mode, but their characteristics suggest the possibility of creating the fused silica capillary walls coating for the separation of anionic and cationic analytes. In the current research, the authors, for the first time, have proposed the approaches to the formation of coatings based on the nanosponges with a molecular weight of 400 and 10 kDa. It was established that the stability of such coatings could be achieved by the introduction of NS into the background electrolyte. The pH range of background electrolytes for the subsequent electrophoretic experiments was determined to be from 4 to 9. The NS-based coatings were compared with the previously investigated coatings based on the crosslinked polystyrene nanoparticles ( NPs ), functionalized with the quaternary ammonium groups. Capillaries modified with NS (400 kDa) were tested for the electrophoretic separations of wide range of analytes: carboxylic acids, aminoacids, fluoroquinoline antibiotics, biogenic amines, and proteins. The separation selectivity of carboxylic acids on the NS-modified capillaries was different from the separation selectivity on the capillaries modified with NPs, functionalized with quaternary ammonium groups. NS affect the separation selectivity of aminoacids by interacting with these analytes, while for fluoroquinoline antibiotics NS act as an agent generating the reversed electroosmotic flow ( EOF ). The modification of fused silica capillary walls by NS with molecular mass of 400 kDa prevented the sorption of biogeniс amines and lyzocyme protein during their electrophoretic separation. Keywords : capillary electrophoresis, nanoparticles, coatings of fused silica capillary walls DOI: http://dx.doi.org/10.15826/analitika.2019.23.2.006 (Russian) D.A. Polikarpova 1 , D.V. Makeeva 1 , L.A. Kartsova 1 , V.A. Davankov 2 , L.A. Pavlova 2 1 Saint-Petersburg State University, Institute of Chemistry, Universitetskii pr. 26., St. Petersburg, Petergof, 198504, Russian Federation 2 A.N. Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St. 28, Moscow, 119334, Russian Federation

Micellar Electrokinetic Chromatography.

Micellar electrokinetic chromatography (MEKC) is a mode of capillary electrophoresis that allows for the separation of neutral molecules in an electric field. Typically, neutral molecules move with electroosmotic flow (EOF) or bulk flow during electrophoretic separations resulting in no temporal resolution between mixtures of neutral analytes. Inclusion of surfactant micelles in the separation buffer allows for the separation of neutral analytes from one another through association with the micelle. Here we outline the implementation of MEKC for the separation of neutral molecules using a mixture of nitroaromatic explosives and their degradation products serving as a test analyte mixture.

CE with multi-walled carbon nanotubes (MWCNTs). Part II. SDS coated functionalized MWCNTs as pseudo-stationary phases in nanoparticle EKC – Retention behaviors of small and large solutes

In this study, functionalized multi-walled carbon nanotubes (MWCNTs), namely hydroxylated MWCNTs (MWCNT-OH), carboxylated MWCNTs (MWCNT-COOH) and sulfonated MWCNTs (MWCNT-SO3H) coated with sodium dodecyl sulfate (SDS) were demonstrated as effective pseudo-stationary phases (PSPs) in the separation of various species by the nanoparticle capillary electrokinetic chromatography (NPEKC) mode of capillary electrophoresis (CE). Due to the significant increase in their surface charge density in the presence of SDS, the three SDS coated MWCNTs yielded high performance separation for herbicides, barbiturates, dansyl-DL-amino acids (Dns-AAs), dipeptides and proteins by NPEKC. In addition, high resolution tryptic peptide maps of three standard proteins including myoglobin, cytochrome C and lysozyme were readily obtained. The three PSPs systems yielded high plate numbers that spanned a wide range of values depending on the type of species. The values of the observed selectivity factors (i.e., α values) were significantly different among the three PSPs for solutes that underwent strong interactions with the SDS coated functionalized MWCNTs while for negatively charged solutes (e.g., Dns-AAs) of the same charge sign as the PSPs the α values were about the same on the three different PSPs indicating weak association with the PSPs and signaling separation based chiefly on the differences in electro-migration arising from differences in charge-to-mass ratios.

Glycoform analysis of alpha1-acid glycoprotein based on capillary electrophoresis and electrophoretic injection

A method based on capillary electrophoresis (CE) with electrophoretic injection and absorbance detection was developed for the direct analysis of AGP glycoforms in human serum. Electrophoretic injection of AGP was performed in the reversed-polarity mode of CE with a capillary coated with poly(ethylene oxide) and that had minimal electroosmotic flow. This situation created an essentially stationary interface between the sample and running buffer during injection and sample stacking. This approach allowed an 11,000-fold increase in sample loading for a 5min injection versus hydrodynamic injection and without introducing any significant levels of extra band-broadening. This method was used with sample pretreatment methods based on acid precipitation and desalting to examine AGP glycoforms in only 65μL of serum. A limit of detection of 2.1–11.3nM was obtained for the major AGP glycoform bands in serum, and the sample pretreatment method gave a recovery of 72.3–80.9% for these glycoforms. The precision for the migration times was ±0.08–0.13% and the precision for the peak areas was ±0.34–1.18% when using serum samples and an internal standard. This method was used for both normal pooled serum and serum from individuals with systemic lupus erythematosus. Results were obtained in a separation time of 25min and allowed the comparison of up to eleven glycoform bands in these samples. A similar approach may be useful in examining additional glycoproteins in serum or other types of biological samples.

A novel enantioseparation approach based on liposome electrokinetic capillary chromatography

As a novel separation mode of capillary electrophoresis (CE), liposome electrokinetic capillary chromatography (LEKC) has aroused considerable attention in recent years; however, the enantioseparation based on this new system has not been previously investigated. In this study, we proposed a brand-new LEKC chiral separation approach using liposomes comprised of phosphatidylcholine (PC) and cholesterol as pseudo-stationary phase and sulfobutyl ether-β-cyclodextrin (SBE-β-CD) as chiral selector. Compared with the single CD system and CD-SDS-MEKC system, this LEKC method presented an obviously preferable enantioseparation of four model drugs (naproxen, warfarin, ketoprofen and amlodipine). In this new established system, all the enantiomers represented baseline separations with the resolution and selectivity respectively achieving 1.584/1.067 (for naproxen), 2.226/1.045 (for warfarin), 1.537/1.038 (for ketoprofen) and 2.592/1.097 (for amlodipine), while other two comparative systems demonstrated no separation or a poor separation. Several important parameters affecting the enantioseparation, such as buffer pH, concentration of liposomes, phosphate buffer solution (PBS) and chiral selector (SBE-β-CD), and applied voltage were systematically investigated. Satisfactory repeatability was achieved through intra-day, inter-day and batch-to-batch investigations with relative standard deviations less than 3.40%. Furthermore, the established method was successfully applied to test the chiral impurity of naproxen sample.

Multiple modes of capillary electrophoresis applied in peptide nucleic acid related study

Peptide Nucleic Acid (PNA) is a nucleic acid analogue, whose neutrally charged and hydrophobic backbone makes it more stable in vivo, so it might act as a potentially better protein probe as compared to aptamer. Currently the investigation of PNA and protein interaction is scarce. In this research, multiple modes of capillary electrophoresis were established and applied for PNA characterization and its interaction with ssDNA and protein. A 15-mer PNA having the same nucleobase sequence as 15-mer anti-thrombin DNA aptamer was chosen as PNA model for this study, its pI (7.71) was estimated by capillary isoelectric focusing (cIEF). Due to its neutral charge and strong hydrophobicity, three micellar electrokinetic chromatography (MEKC) modes containing (a) SDS, (b) Triton X-100 and (c) CTAB were compared for PNA related analysis. CTAB was not applicable for PNA analysis, while in 4mM SDS or 2mM Triton X-100, PNA and PNA-ssDNA complex can be identified directly. The significant peak of PNA-ssDNA complex helped in validating the two MEKC modes for PNA and target interaction study. Furthermore, the effect of SDS and Triton X-100 concentrations in the two MEKC modes on the protein target thrombin analysis was investigated by capillary zone electrophoresis (CZE). 4mM SDS caused thrombin denaturation. So in 2mM Triton X-100, interactions of PNA with thrombin, PNA with RNase A and a non-aptameric PNA (n-PNA) with thrombin were compared. PNA with thrombin exhibited strongest binding. In summary, cIEF mode for PNA pI determination, MECK mode for direct PNA, PNA-ssDNA and PNA-protein complex identification and CZE mode for the effect of surfactant in MEKC modes on protein target thrombin analysis were applied. Above results showed that multiple modes of CE provide rapid and very low-sample cost methods for PNA related studies.

Plug-plug kinetic capillary electrophoresis for in-capillary exoglycosidase digestion as a profiling tool for the analysis of glycoprotein glycans

An online exoglycosidase digestion was combined with a plug-plug kinetic mode of capillary electrophoresis (CE) for the analysis of glycoprotein-derived oligosaccharides. An exoglycosidase solution and a solution of glycoprotein glycans derivatized with 8-aminopyrene-1,3,6-trisulfonic acid (APTS) were introduced to a neutrally coated capillary previously filled with electrophoresis buffer solution containing 0.5w/v% hydroxypropylcellulose. After immersion of both ends of the capillary in the buffer solutions, a negative voltage was applied for analysis. An APTS group of an oligosaccharide derivative has triply negative charges, which forced saccharide derivatives to anode with fast mobility and pass through the enzyme plug, which are detected at the anodic end. If the terminal monosaccharides of APTS-labeled oligosaccharides are released by the action of an exoglycosidase, the migration times of the oligosaccharides shift to those of digested oligosaccharides. We examined β-galactosidase, α-mannosidase, β-N-acetylhexosaminidase, α-neuraminidase, and α-fucosidase, and found only β-galactosidase and α-neuraminidase showed good reactivity toward APTS-labeled oligosaccharides; the reaction was completed by injecting a 3.6cm long plug of 200 and 50mU/mL concentration of exoglycosidases. In contrast, other exoglycosidases could not react with APTS labeled oligosaccharides at a concentration up to 5U/mL. The β-N-acetylhexosaminidase reaction was successively followed by the electrophoretic mobility of APTS oligosaccharides and stopped for 10min when saccharide derivatives were achieved in the enzyme plug. The reaction of α-fucosidase and α-mannosidase was completed by decreasing the electrophoretic voltage to −2kV when the APTS oligosaccharides were passing through an exoglycosidase plug. We established the CE conditions for all of the glycosidic linkage analysis of glycoprotein glycans.