Capillary Electrophoresis Profiles Research Articles

Focus on the Protein Fraction of Sports Nutrition Supplements.

Increasing awareness of balanced diet benefits is boosting the demand for high-protein food and beverages. Sports supplements are often preferred over traditional protein sources to meet the appropriate dietary intake since they are widely available on the market as stable ready-to-eat products. However, the protein components may vary depending on both sources and processing conditions. The protein fraction of five commercial sports supplements was characterized and compared with that of typical industrial ingredients, i.e., whey protein concentrates and isolates and whey powder. The capillary electrophoresis profiles and the amino acid patterns indicated that, in some cases, the protein was extensively glycosylated and the supplemented amino acids did not correspond to those declared on the label by manufacturers. The evaluation by confocal laser scanning microscopy evidenced the presence of large aggregates mainly enforced by covalent crosslinks. The obtained findings suggest that, beside composition figures, provisions regarding sports supplements should also consider quality aspects, and mandatory batch testing of these products would provide more reliable information to sport dieticians.

Development and application of an analytical approach to assess an antibody's potential for disulfide reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction has been observed in a recent large-scale clinical manufacturing operation. A massive reduction/precipitation at post-clarification steps has occurred. This note presents the development of a novel analytical approach to identify the "potential reduction"-a unique approach to predict the propensity of a monomeric-profiled mAb to be reduced in the post-harvest stage, such as harvest clarification and/or purification steps. The core of this new approach includes comparing the non-reducing capillary electrophoresis profiles of pre- and post-vacuum treated mAb in harvest cell culture fluid (HCCF). Using this approach, the potential reductions of two in-house mAbs in the unclarified and clarified cell culture harvest were assessed.

Validation of a neural network approach for STR typing to replace human reading

A typical forensic laboratory process for interpreting STR capillary electrophoresis profile data is for two people to independently ‘read’ the profiles, compare results, and resolve any differences. Recently, work has been conducted to develop a machine learning tool called an artificial neural network (ANN) to carry out the same function as a human profile reader, by classifying areas of fluorescence in the capillary electrophoresis profile raw signal data. The ANN approach has been embedded in commercial software FaSTR™ DNA to read GlobalFiler™ DNA profiles. The ANN feature of FaSTR™ DNA was investigated during validation at Forensic Science South Australia (FSSA) to determine whether one of the human profile readers could be replaced by an ANN reader. FaSTR™ DNA accuracy in detecting allele peaks in reference profiles was 99.7% and was deemed high enough that a one-reader workflow could be implemented into the reference reading workflow at FSSA.

Electrophoretic Profile of Serum Proteins Using Capillary Technique in Patients Attending the Douala General Hospital, Cameroon

Background: Electrophoresis of serum proteins is an orientation examination routinely used in clinical practice. For a few years, agarose gel electrophoresis has tended to be replaced with capillary electrophoresis owing to an increase in the accuracy of results. However, this technique is uncommon and is not widely used in Cameroon. Objectives: The research aimed at studying the electrophoretic profile of serum proteins using capillary technique among patients attending the Douala General Hospital, Cameroon. Methods: Capillary electrophoresis was used to carry out tests on blood samples from any inpatients and outpatients and fasting for 8-12 hours. Capillary electrophoresis of serum samples was used for the separation of proteins into six fractions and the total protidemia of each serum samples was determined using the Biuret method. Results were interpreted by observing the shape of curves and quantitative variations in each fraction of the different serum proteins. Results: A total of 311 patients participated in the study. The sampled population aged 50±18 years on average and consisted of 55.3% men and 44.7% women. All capillary electrophoresis profiles presented six protein fractions, namely, albumin, alpha (1 and 2), beta (1 and 2) and gamma globulins. Pathological disorders were diagnosed in 290 patients and 21 patients had normal results. Inflammatory syndromes accounted for 63.34% and monoclonal gammopathies for 10.29% the main pathological disorder identified. Conclusion: Capillary electrophoresis provides a more precise identification of biological syndromes and clear distinction of the six fractions of each protein. Monoclonal profiles and inflammatory syndromes were well detected. A prevalence of 10.29% was determined for gammopathies.

Investigation of Sequence Clipping and Structural Heterogeneity of an HIV Broadly Neutralizing Antibody by a Comprehensive LC-MS Analysis.

CAP256 is one of the highly potent, broadly neutralizing monoclonal antibodies (bNAb) designed for HIV-1 therapy. During the process development of one of the constructs, an unexpected product-related impurity was observed via microfluidics gel electrophoresis. A panel of complementary LC-MS analyses was applied for the comprehensive characterization of CAP256 which included the analysis of the intact and reduced protein, the middle-up approach, and a set of complementary peptide mapping techniques and verification of the disulfide bonds. The designed workflow allowed to identify a clip within a protruding acidic loop in the CDR-H3 region of the heavy chain, which can lead to the decrease of bNAb potency. This characterization explained the origin of the additional species reflected by the reducing gel profile. An intra-loop disulfide bond linking the two fragments was identified, which explained why the non-reducing capillary electrophoresis (CE) profile was not affected. The extensive characterization of CAP256 post-translational modifications was performed to investigate a possible cause of CE profile complexity and to illustrate other structural details related to this molecule's biological function. Two sites of the engineered Tyr sulfation were verified in the antigen-binding loop, and pyroglutamate formation was used as a tool for monitoring the extent of antibody clipping. Overall, the comprehensive LC-MS study was crucial to (1) identify the impurity as sequence clipping, (2) pinpoint the clipping location and justify its susceptibility relative to the molecular structure, (3) lead to an upstream process optimization to mitigate product quality risk, and (4) ultimately re-engineer the sequence to be clip-resistant. Graphical Abstract ᅟ.

Automated band annotation for RNA structure probing experiments with numerous capillary electrophoresis profiles.

Capillary electrophoresis (CE) is a powerful approach for structural analysis of nucleic acids, with recent high-throughput variants enabling three-dimensional RNA modeling and the discovery of new rules for RNA structure design. Among the steps composing CE analysis, the process of finding each band in an electrophoretic trace and mapping it to a position in the nucleic acid sequence has required significant manual inspection and remains the most time-consuming and error-prone step. The few available tools seeking to automate this band annotation have achieved limited accuracy and have not taken advantage of information across dozens of profiles routinely acquired in high-throughput measurements. We present a dynamic-programming-based approach to automate band annotation for high-throughput capillary electrophoresis. The approach is uniquely able to define and optimize a robust target function that takes into account multiple CE profiles (sequencing ladders, different chemical probes, different mutants) collected for the RNA. Over a large benchmark of multi-profile datasets for biological RNAs and designed RNAs from the EteRNA project, the method outperforms prior tools (QuSHAPE and FAST) significantly in terms of accuracy compared with gold-standard manual annotations. The amount of computation required is reasonable at a few seconds per dataset. We also introduce an 'E-score' metric to automatically assess the reliability of the band annotation and show it to be practically useful in flagging uncertainties in band annotation for further inspection. The implementation of the proposed algorithm is included in the HiTRACE software, freely available as an online server and for download at http://hitrace.stanford.edu. sryoon@snu.ac.kr or rhiju@stanford.edu Supplementary data are available at Bioinformatics online.

Nucleotide Polymorphism-Based Single-Tube Test for Robust Molecular Identification of All Currently Described Brucella Species

Among the numerous molecular methods described during the last 20 years to identify Brucella, multiplexed amplification methods offer the cheapest and simplest technical solution for molecular identification. However, one disadvantage of such methods is their need to undergo technical revalidation each time a new marker is added to the system. Moreover, polymorphic markers cannot be assessed at the single-nucleotide level in these assays. Since new Brucella species are continuously being described, open methodologies able to accommodate new markers while preserving all other system parameters have an obvious advantage. We present a ligase chain reaction (LCR)-based method that simultaneously assesses multiple genetic markers at the single-nucleotide level. Most of the selected markers originate from a multilocus sequence typing (MLST) database that has been extensively validated on hundreds of different Brucella strains. When assayed on both reference and field strains, the method yields characteristic capillary electrophoresis profiles for each of the 10 Brucella species described to date and displays discriminatory potential below the species level for some. Since the LCR methodology is insensitive to interference resulting from the use of multiple oligonucleotides in a single mixture, the way is open for smooth future updates of the proposed system. Such updates are inevitable, given the pending description of new Brucella species.

HiTRACE: high-throughput robust analysis for capillary electrophoresis

Capillary electrophoresis (CE) of nucleic acids is a workhorse technology underlying high-throughput genome analysis and large-scale chemical mapping for nucleic acid structural inference. Despite the wide availability of CE-based instruments, there remain challenges in leveraging their full power for quantitative analysis of RNA and DNA structure, thermodynamics and kinetics. In particular, the slow rate and poor automation of available analysis tools have bottlenecked a new generation of studies involving hundreds of CE profiles per experiment. We propose a computational method called high-throughput robust analysis for capillary electrophoresis (HiTRACE) to automate the key tasks in large-scale nucleic acid CE analysis, including the profile alignment that has heretofore been a rate-limiting step in the highest throughput experiments. We illustrate the application of HiTRACE on 13 datasets representing 4 different RNAs, 3 chemical modification strategies and up to 480 single mutant variants; the largest datasets each include 87 360 bands. By applying a series of robust dynamic programming algorithms, HiTRACE outperforms prior tools in terms of alignment and fitting quality, as assessed by measures including the correlation between quantified band intensities between replicate datasets. Furthermore, while the smallest of these datasets required 7-10 h of manual intervention using prior approaches, HiTRACE quantitation of even the largest datasets herein was achieved in 3-12 min. The HiTRACE method, therefore, resolves a critical barrier to the efficient and accurate analysis of nucleic acid structure in experiments involving tens of thousands of electrophoretic bands.

Study of the capillary electrophoresis profile of intact α-1-acid glycoprotein isoforms as a biomarker of atherothrombosis

α-1-Acid glycoprotein (AGP) is a serum glycoprotein that presents several isoforms. Changes in the isoforms of AGP have been related to different pathological states including cardiovascular diseases (CVDs) such as acute myocardial infarction. However, to our knowledge, the role of variations of AGP isoforms as a potential biomarker of atherothrombosis has not been addressed. In this work, a preliminary study about differences in the capillary zone electrophoresis (CZE) profile of intact (non-hydrolyzed) AGP isoforms between healthy individuals and patients with atherothrombosis, specifically abdominal aortic aneurysm (AAA) and carotid atherosclerosis (CTA), has been performed. Biological samples (plasmas and sera) were analyzed by CZE after immunoaffinity chromatography purification. Up to 13 peaks corresponding to groups of isoforms of intact AGP from plasma samples were detected by CZE-UV. Electrophoretic profiles were aligned, peaks assigned, and linear discriminant analysis (LDA) of percentage of the corrected areas of AGP peaks was employed to discriminate and classify the CZE profiles of AGP samples. LDA enabled to accomplish 92.9% of correct classification of the AGP samples when the three groups of samples were considered. Besides, the LDA model showed high predictive power in the groups healthy vs. sick, healthy vs. AAA, and healthy vs. CTA. The described method was a successful approach to study the potential of AGP isoforms profile as a biomarker of atherothrombosis. To the best of our knowledge this has been the first time that a possible role of the CZE profile of intact AGP isoforms as a biomarker of vascular diseases has been demonstrated.

Capillary electrophoresis profiles and fluorophore components of humic acids in Nebraska corn and Philippine rice soils

As humic substances represent relatively high molecular mass polyelectrolytes containing aromatic, aliphatic and heterocyclic subunits, capillary electrophoresis (CE) has become an attractive method for “finger-print” characterization of humic acids. In addition, fluorescence excitation-emission matrix (EEM) spectroscopy is also a highly sensitive analytical method, providing information on flurophore properties of humic acids. However, basic information on the CE profile patterns and fluorophore components of humic acid fractions from different agricultural soils is rare. Consequently, we sequentially extracted the mobile humic acid (MHA) and recalcitrant Ca humate (CaHA) fractions from Nebraska corn soils and Philippine rice soils under different fertilization schemes, and acquired their CE profiles and fluorophore component composition. We observed greater differences in the CE profiles between soils than between management practices on the same soil, reflecting the distinct “finger-print” CE features of these soil humic fractions. Modeling analysis of EEM data indicated that four fluorophore components were present in these humic fractions. Whereas three have previously been reported in literature, we propose the features of the unknown component as being relevant to agricultural humic fractions. UV irradiation seemed to impact the Nebraska soils by disaggregating the humic fractions into their structural subunits, while impacting the Philippine soils by altering the functional groups of humic fractions. This research provides insight into the CE profiles and fluorophore features of humic acid fractions from different agricultural soils.

A polymerase chain reaction-coupled high-resolution melting curve analytical approach for the monitoring of monospecificity of avian Eimeria species

Coccidiosis is a significant disease of poultry caused by different species of Eimeria. Differentiation of Eimeria species is important for the quality control of the live attenuated Eimeria vaccines derived from monospecific lines of Eimeria spp. In this study, high-resolution melting (HRM) curve analysis of the amplicons generated from the second internal transcribed spacer of nuclear ribosomal DNA (ITS-2) was used to distinguish between seven pathogenic Eimeria species of chickens, and the results were compared with those obtained from the previously described technique, capillary electrophoresis. Using a series of known monospecific lines of Eimeria species, HRM curve analysis was shown to distinguish between Eimeria acervulina, Eimeria brunetti, Eimeria maxima, Eimeria mitis, Eimeria necatrix, Eimeria praecox and Eimeria tenella. Computerized analysis of the HRM curves and capillary electrophoresis profiles could detect the dominant species in several specimens containing different ratios of E. necatrix and E. maxima and of E. tenella and E. acervulina. The HRM curve analysis identified all of the mixtures as “variation” to the reference species, and also identified the minor species in some mixtures. Computerized HRM curve analysis also detected impurities in 21 possible different combinations of the seven Eimeria species. The PCR-based HRM curve analysis of the ITS-2 provides a powerful tool for the detection and identification of pure Eimeria species. The HRM curve analysis could also be used as a rapid tool in the quality assurance of Eimeria vaccine production to confirm the purity of the monospecific cell lines. The HRM curve analysis is rapid and reliable and can be performed in a single test tube in less than 3 h.

Capillary electrophoresis–mass spectrometry as a characterization tool for therapeutic proteins

With the increasing use of capillary electrophoresis (CE) in the biotechnology industry, there is a demand for analytical tools and methodology that can be used to characterize CE profiles. This article describes the implementation and optimization of a robust online CE–mass spectrometry (CE–MS) system used for the characterization of several CE assays developed at Genentech Inc. These assays include CE as a complement to reverse-phase peptide mapping for the identification of small peptides eluting in the void volume, profiling N-linked glycopeptide heterogeneity, and determining O-linked site occupancy. In addition, CE–MS was used to confirm major 8-aminopyrene-1,3,6-trisulfonate (APTS)-labeled glycans released from recombinant antibodies that are routinely profiled by CE–laser-induced fluorescence (CE–LIF). For each study, CE–MS was able to successfully identify components seen in UV or LIF electropherograms, thereby expanding the capability of CE and CE–MS for profiling biomolecules.

Detection of Structural Change of Superoxide Dismutase in Solution

We have confirmed that dissociation of the dimeric SOD molecule into a monomeric one can be readily detected in solution by the use of capillary electrophoresis (CE), which is based on the fact that the peak height in the CE profile is highly dependent on the aggregation conditions of the protein molecule. Based on this fact, it has become apparent that the hydrogen peroxide molecule induces the dissociation of the dimeric structure of SOD, and this should give reasonable explanation for the inactivation of SOD by hydrogen peroxide. Our results may give a convenient way for the early detection of the amyotrophic lateral sclerosis in patients, because we can estimate whether the SOD molecule is of a rigid or loosed dimeric structure by the use of this technique. The loosed one has been assumed to exhibit inherent toxicity of the copper center, so-called "gain-of-function" of the mutant SOD.

Clotting and proteolytic properties of plant coagulants in regular and ultrafiltered bovine skim milk

Regular and ultrafiltered (UF; 1×, 2× and 4× concentrated) skim milk samples were treated with a range of enzymes including calf rennet, ficin and papain. The clotting properties, curd casein profiles and free amino acid (FAA) contents were determined. In general, UF milk samples coagulated faster and formed firmer curds irrespective of protein concentration. Furthermore, both ficin and papain had a more significant effect on proteolysis in curd formed from regular and 1× UF milk than on 2× or 4× UF milk. Cardoon extract and calf rennet had very similar clotting properties, although the former caused both the capillary electrophoresis profile of caseins and FAA measurements to show slightly more extensive hydrolysis in the curd. The results suggest that the UF process may cause structural changes to proteins or other milk constituents with a resultant change in clotting properties and proteolysis of the casein molecules.

Distinction of water-soluble constituents between natural and cultured Cordyceps by capillary electrophoresis

Cordyceps is an expensive traditional Chinese medicine, which has anti-tumor activity and significant effects on the immune system. In Southeast Asia, Cordyceps is commonly sold in capsule form as a health food product. Most of these products are derived from cultured Cordyceps mycelia. Because of the price difference, some manufacturers claim their products are from natural Cordyceps. In order to distinguish among various types of Cordyceps in the market, the profiles of water-soluble constituents derived from different sources of Cordyceps were determined by capillary electrophoresis (CE). Both natural and cultured Cordyceps showed three peak clusters migrated at 5-7, 9-11 and 12-13 min, and the height and resolution of these peak clusters were rather distinct. Peak cluster at 9-11 min was identified as adenosine, guanosine and uridine, and shared a similarity between natural and cultured products. In contrast, the peak cluster at 5-7 min was characteristic of natural Cordyceps, regardless of hosts and sources. By using the peak characteristics of CE profiles of different Cordyceps samples, hierarchical clustering analysis was performed. The result shows that those samples of natural Cordyceps were grouped together distinct from the cultured and commercial products. Thus, the CE profiles could serve as fingerprints for the quality control of Cordyceps.

Simultaneous determination of six main nucleosides and bases in natural and cultured Cordyceps by capillary electrophoresis

A simple method is described for simultaneous determination of six main nucleosides and bases including adenine, uracil, adenosine, guanosine, uridine and inosine in Cordyceps by capillary electrophoresis (CE). Chemometric optimization based on central composite design was employed to find the optimum resolution. The optimum factor space was defined by three parameters: buffer concentration, pH and concentration of acetonitrile as organic modifier. Resolution ( R S) was employed to evaluate the response function. A running buffer composed of 500 mM boric acid, adjusted pH to 8.6 with sodium hydroxide and 12.2% acetonitrile as modifier was found to be the most appropriate for the separation. The contents of the six components were determined by using adenosine monophosphate as an internal standard. Furthermore, hierarchical clustering analysis based on characteristics of 32 peaks in CE profiles from the tested 12 samples showed that natural and cultured Cordyceps were in different clusters. Adenosine and inosine were extracted as markers for discrimination of natural Cordyceps. The result of clustering based on the two peaks characteristics was in excellent agreement with that based on 32 peaks’. Thus, adenosine and inosine could be used as markers for quality control of natural and cultured Cordyceps.

Hydrolysis, lactonization, and identification of alpha(2 --> 8)/alpha(2 --> 9) alternatively linked tri-, tetra-, and polysialic acids.

Alpha-(2 --> 8)/alpha(2 --> 9) alternatively linked polysialic acid (PSA) can be identified by controlled hydrolysis followed by the analysis with capillary electrophoresis (CE). Due to the different stability of alpha(2 --> 8) and alpha(2 --> 9) linkages in acidic hydrolysis, oligosialic acids (OSAs) from the hydrolysis of alpha(2 --> 8)/alpha(2 --> 9) OSA/PSA could be classified into two groups in the CE profile. The group with an odd numerical degree of polymerization (DP) had two peaks in the CE profile, and the other group, with even number of DP, showed one peak. Each alternating alpha(2 --> 8)/alpha(2 --> 9) linked OSA contains two isomers: one starts with the alpha(2 --> 8) linkage from the nonreducing end and the other starts with the alpha(2 --> 9) linkage from the nonreducing end. Trimers and tetramers were isolated by using a Mono Q column with an HPLC system. The two trimer isomers are alpha(2 --> 8)/alpha(2 --> 9) and alpha(2 --> 9)/alpha(2 --> 8) linkages and only showed partial separation by CE. After lactonization, sialidase hydrolysis, and alkaline treatment, the two trimer isomers could be separated and identified by CE analysis, but only the alpha(2 --> 8)/alpha(2 --> 9) trimer could be converted to the dilactone in glacial acetic acid. The two tetramer isomers could be converted to four monolactones and three dilactones. These lactonized species could be identified on the basis of several principles in sialidase hydrolysis and lactonization. In conclusion, regioselectivity on the lactonization of oligosialic acids proceeds under several principles: (1) Lactonization takes place more easily in the alpha(2 --> 8) linkage than in the alpha(2 --> 9) linkage; (2) all of the positions of alpha(2 --> 8) linkages in alpha(2 --> 8)/alpha(2 --> 9) alternatively linked OSA can be lactonized regardless of external or internal carboxyl groups involved; and (3) for the site of alpha(2 --> 9) linkage, only internal carboxyl groups can be lactonized.

Capillary electrophoresis profiles of fucoidan and heparin fractions: significance of mobility dispersity for their characterization

AbstractCapillary electrophoresis was investigated for characterization of the natural anionic polysaccharide fucoidan from marine brown algae and its comparison with heparin under different pH, ionic strength, and counter‐ion conditions. Reproducible profiles of these polysaccharides were obtained in indirect UV detection mode by introducing sulfosalicylic acid or anisic acid as chromophores in background electrolytes. Theoretical evaluations of zone spreading originating from molecular diffusion, electromigration, and initial zone width have shown that these dispersive contributions remain negligible compared to the overall profile dispersion. Therefore, the conversion of the experimental electropherograms (absorbance vs. time plots) into absorbance vs. electrophoretic mobility curves enables a comparison of these polysaccharides in terms of electrophoretic mobility dispersity. Nevertheless, a linear correlation between mobility and chemical charge rate cannot be established for these highly sulfated polysaccharides, because their chemical charge rate is higher than the threshold value for which counter‐ion condensation occurs.

Differences in capillary electrophoresis profiles of urinary and recombinant erythropoietin.

Different profiles were obtained by capillary zone electrophoresis (CZE) of human erythropoietin (EPO) of recombinant and urinary origin. To unambiguously detect doping by EPO, direct methods able to determine the presence of the drug itself in a physiological fluid are required. Since the host cell line used for EPO production influences its glycosylation, the carbohydrate distribution of natural human EPO may be different from that of recombinant EPO. The different content in sialic acid groups between recombinant and endogenous EPO provide a basis for their distinction by CZE.

Mass spectrometry-based procedure for the identification of ovine casein heterogeneity.

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HTPC into four major peaks. With ESI-MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography-ESI-MS allowed us to determine each fraction's composition by detecting thirteen alphas1-, eleven alphas2-, seven beta-, and three kappa-casein (CN) components. The alphas1-CN and alphas2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as kappa-CN and beta-CN. By CE at pH 2.5, each casein fraction was as heterogeneous as that resulting from ESI-MS for the single HPLC-derived fractions. The separation of alphas1-CN and alphas2-CN proved to be excellent, with the exception of a co-migration of kappa0-CN with a minor alphas1-CN component and of a glycosylated kappa-CN for with low-phosphorylated = alphas1-CN and beta-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI-MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative alphas1-CN variants, the non-allelic alphas1-CN and alphas2-CN forms, dimeric kappa-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.