Capillary Transient Isotachophoresis Research Articles

Determination of 90Sr in highly radioactive aqueous samples via conversion to a kinetically stable 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex followed by concentration–separation–fractionation based on capillary electrophoresis–liquid scintillation

BackgroundThe Fukushima Daiichi Nuclear Power Plant accident (2011) released large amounts of radioactive substances into the environment and generated highly radioactive debris. Post-accident countermeasures are currently in the phase of fuel debris removal, which requires the analysis of radioactive contaminants in the environment and fuel. The spectra of solely β-emitting nuclides, such as 90Sr, overlap; thus, an effective method for nuclide separation is desired. Since conventional methods for high-dose sample analysis pose substantial exposure risks and generate large amounts of secondary radioactive waste, faster procedures allowing for decreased radiation emission are highly desirable. ResultsIn this study, we developed a 90Sr2+ quantitation technique based on liquid scintillation counting (LSC)–coupled capillary transient isotachophoresis (ctITP), along with two-point detection and relying on the rapid concentration, separation, and fractionation of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-complexed 90Sr2+ in a single run. The applicability of our method for the analysis of real-world samples was verified by conducting addition-recovery experiments using a seawater reference material and radioactive liquid waste obtained from the radioactive waste treatment facility at the Japan Atomic Energy Agency. The recovery determined by LSC was 95–113%, indicating successful quantitative analysis. 90Sr recovery was determined to be 90.1% from a contaminated water sample obtained from the Fukushima Daiichi Nuclear Power Plant, which was analyzed using the standard addition of 90Sr. The sensitivity (detection limit = 0.016 Bq) of the proposed method on a radioactivity basis was equal to or higher than that of the conventional method using ion exchange-LSC (0.012–0.07 Bq). Significance and noveltyOur method allows for the handling of high-dose radioactive samples at the microliter level and is substantially faster than conventional ion exchange protocols, whereas ctITP has not been used for practical applications due to inaccurate collection and lack of a suitable chemical system. The concentration–separation–fractionation protocol in ctITP is successful due to the existence of a rare inert Sr2+ complex and precise fractionation. This study establishes a pathway toward safer and more practical analysis of radionuclides.

Analysis of DNA Origami Nanostructures Using Capillary Electrophoresis.

DNA origami nanostructures are engineered nanomaterials (ENMs) that possess significant customizability, biocompatibility, and tunable structural and functional properties, making them potentially useful materials in fields, such as medicine, biocomputing, biomedical engineering, and measurement science. Despite the potential of DNA origami as a functional nanomaterial, a major barrier to its applicability is the difficulty associated with obtaining pure, well-folded structures. Therefore, rapid methods of analysis to ensure purity are needed to support the rapid development of this class of nanomaterials. Here, we present the development of capillary electrophoresis (CE) as an analytical tool for DNA origami. CE was investigated under both capillary zone electrophoresis (CZE) and capillary transient isotachophoresis (ctITP) modes. Optimization of both systems yielded baseline resolved separations of folded DNA origami nanostructures from excess staple strands. The ctITP separation mode demonstrated superior performance in terms of peak resolution (Rs = 2.05 ± 0.3), peak efficiency (N = 12,200 ± 230), and peak symmetry (As = 1.29 ± 0.032). The SYBR family dyes (Gold, Green I, and Green II) were investigated as highly efficient, noncovalent fluorophores for on-column labeling of DNA origami and detection using laser-induced fluorescence. Finally, ctITP analysis conditions were also applied to DNA origami nanostructures with different shapes and for the differentiation of DNA origami aggregates.

Bottom-Up Analysis of Proteins by Peptide Mass Fingerprinting with tCITP-CZE-ESI-TOF MS After Tryptic Digest.

The identification of proteins in samples of moderate to complex composition is primarily done by bottom-up approaches. Therefore, proteins are enzymatically digested, mostly by trypsin, and the resulting peptides are then separated prior to their transfer to a mass spectrometer. The following protocol portrays a bottom-up method, which was optimized for the application of CZE-ESI-TOF MS. Protein denaturation is achieved by addition of 2,2,2-trifluoroethanol (TFE) and heat treatment. Afterwards, disulfide bonds are reduced with tris-(2-carboxyethyl)phosphine (TCEP) and subsequently alkylated with iodoacetamide (IAA). The tryptic digest is performed in an ammonium bicarbonate buffer at pH8.0. The digested protein sample is then concentrated in-capillary by transient capillary isotachophoresis (tCITP) with subsequent CZE separation of tryptic peptides in an acidic background electrolyte. Hyphenation to a time-of-flight (TOF) mass spectrometer is carried out by a triple-tube coaxial sheath flow interface, which uses electrospray ionization (ESI). Peptide identification is done by peptide mass fingerprinting (PMF). The protocol is outlined exemplarily for a model protein, i.e., bovine β-lactoglobulin A.

Capillary electrophoresis-based methodology for screening of oligonucleotide aptamers.

As a new molecular recognition element, oligonucleotide aptamer not only has higher affinity and specificity to target molecules, but also has the advantages of wide recognition range, in vitro synthesis and chemical stability compared with conventional antibodies. Since a kind of screening method termed systematic evolution of ligands by exponential enrichment (SELEX) was reported, scientists have extensively researched the methodology of how to highly and efficiently screen out aptamers from a library consisting of a large number of random oligonucleotides. Certainly capillary electrophoresis-based screening methodologies, including nonequilibrium capillary electrophoresis of equilibrium mixtures, equilibrium capillary electrophoresis of equilibrium mixtures, non-SELEX, ideal-filter capillary electrophoresis, capillary transient isotachophoresis, etc., are revolutionary. Compared with conventional SELEX, these capillary electrophoresis-based methodologies show incomparable advantages such as the single-round screening of aptamers and increased successful screening rate. Methodology studies on the screening process of aptamers are comprehensively reviewed.

Nitrogen-doped carbon dots aid in the separation of ssDNA molecules of different length by capillary transient isotachophoresis (ctITP) with laser-induced fluorescence (LIF) detection

This study demonstrates a novel application of nitrogen-doped carbon dots (NCDs) to enable the separation of different lengths of single-stranded DNA (ssDNA) by eletrokinetic means. Carbon dots have recently found widespread application in the fields of sensing, diagnostics, and healthcare due to their biocompatibility and low toxicity. In light of growing interest in the use of ssDNA aptamers over antibodies in the fields of biosensor development and drug delivery, it is important to establish a simple and effective method for aptamer separation. In this study, we employed NCDs as buffer additives in a capillary electrophoresis (CE)-based method, giving rise to the separation of FAM-labeled ssDNA samples ranging from 32 to 100 bases in length, with resolutions ranging from 1.30 – 1.77. In particular, we adopted a capillary transient isotachophoresis (ctITP) system with laser-induced fluorescence (LIF) detection, with both the separation and sample buffers modified by the addition of 30 μg/mL NCDs. These nanomaterials were prepared by a simple hydrothermal method from a mixture of citric acid and ethylenediamine. The NCDs themselves are highly fluorescent and photostable. As components in the background electrolyte, they did not interfere with the fluorescence emission of the FAM-labeled DNA samples. Under the conditions employed, no separation could be achieved in the absence of the NCDs nor with undoped CDs. The results show that NCDs function as buffer additives capable of enhancing electrokinetic-based separations of ssDNA, and hence, provide a new application for these carbon nanomaterials.

On a separation voltage polarity switching transient capillary isotachophoresis method for higher sample loading capacity and better separation performance.

Limited sample loading capacity is one of the major reasons that prevents the utility of capillary electrophoresis (CE) as a routine separation method as compared to liquid chromatography (LC). In our previous study, separation voltage polarity switching transient capillary isotachophoresis (PS-tCITP) was proposed. Both sample loading capacity and separation resolution could be improved using a single PS-tCITP instead of routine transient capillary isotachophoresis (tCITP). In this study, a detailed investigation on the optimization strategy of the PS-tCITP method was performed systematically. A possible mechanism of sample preconcentration in multiple PS-tCITP was first proposed to better understand the multiple PS-tCITP process. Several optimization experiments were then performed, including single PS-tCITP, paused PS-tCITP and multiple PS-tCITP, sequentially using a mixture of five peptides. By selecting an optimum polarity switching time, sample loading capacity of 100% capillary volume could be achieved in a single PS-tCITP. Introducing an additional pause between each polarity switching in a single PS-tCITP further improved the separation resolution. Experimental results showed a baseline separation of five selected peptide standards at 100% sample loading volume using a 100 min pause in a single PS-tCITP. To further improve separation efficiency while still maintaining 100% sample loading volume, a multiple PS-tCITP technique was developed through this study. Compared to the separation performance of the optimal single PS-tCITP at 100% sample loading volume with a 10 min pause, the separation window was improved by 54% and the peak capacity was improved by 48% in the optimal four PS-tCITP with the same sample loading volume and pause.

Front cover: Purification of anionic fluorescent probes through precise fraction collection with a two‐point detection system using multiple‐stacking preparative capillary transient isotachophoresis

Front cover: Purification of anionic fluorescent probes through precise fraction collection with a two‐point detection system using multiple‐stacking preparative capillary transient isotachophoresis

Purification of anionic fluorescent probes through precise fraction collection with a two-point detection system using multiple-stacking preparative capillary transient isotachophoresis.

A novel combination of CE-based separation techniques was used for the precise fractionation of ionic compounds from impurities. The combination of on-capillary concentration and separation using transient isotachophoresis, with multiple injections and a two-point detection system provided higher efficiency, and accuracy at a microliter-scale injection volume, than when CE was individually used for purification. In this paper, we present successful applications of the CE fractionation techniques for the purification of fluorescein, fluorescein-4-isothiocyanate, two fluorescent metal ion probes, and a fluorescein-modified DNA aptamer. The purity of the isolated fluorescent probes ranged from 95 to 99%. Such high purity could not be achieved using chromatographic purification techniques. With relatively low dilution factors of 6-9, the purified probe solutions were practical for use as purified stock solutions. In addition, the fluorescein-modified DNA aptamer purified by our method was successfully used in a thrombin binding assay. The method developed was useful for the purification of anionic fluorescent reagents to be of ultratrace analytical grade for use with CE-LIF.

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.

A separation voltage polarity switching method for higher sample loading capacity and better separation resolution in transient capillary isotachophoresis separation.

A separation voltage polarity switching transient capillary isotachophoresis (PS-tCITP) was developed to overcome a major sample loading volume limitation in transient capillary isotachophoresis (tCITP). The fundamental idea of PS-tCITP is to let sample ions move back and forth in a separation capillary during their initial isotachophoresis focusing stage by switching the polarity of the separation voltage, in order to both increase the sample loading volume and improve the separation efficiency as compared to the conventional tCITP method. The experimental evaluation of the novel PS-tCITP method by using two peptide standards at 2 μM concentration showed that the maximum sample loading volume could be increased from 45% of the total separation capillary volume in tCITP to 70% in PS-tCITP, which resulted in a more than 1.5 fold increase in the peptide peak intensity at a given length/volume of the separation capillary. Due to the consecutive focusing of sample volume from each polarity switching of the separation voltage, the separation time window at a given sample loading volume was also increased significantly in PS-tCITP as compared to tCITP. Experiment comparison between tCITP and PS-tCITP at 45% sample loading volume using the same setup showed that the migration time difference between the two peptide peaks increased from 0.3 min in tCITP to 0.363 min in PS-tCITP with similar peak widths and heights, resulting in roughly a 21% improvement in separation resolution. The performance advantages of PS-tCITP separation over tCITP separation were further verified by using a mixture of six peptide standards.

Monitoring of Deamidation and Lanthionine Formation in Recombinant Mugwort Allergen by Capillary Zone Electrophoresis (CZE)-UV and Transient Capillary Isotachophoresis-CZE-Electrospray Ionization-TOF-MS.

The response to thermal stress is an important parameter relevant for characterizing the biological activity and long-term stability of recombinant proteins, which may show irreversible, pH dependent structural changes under these conditions. We selected the recombinant pollen allergen of mugwort ( Artemisia vulgaris) rArt v 3.0201 as a relevant model to study structural changes due to thermal and pH stress by means of capillary zone electrophoresis (CZE)-UV and capillary zone electrophoresis (CZE)-electrospray ionization (ESI)-TOF-MS. Therefore, this recombinant protein was exposed to 95 °C under acidic (pH 3.4) and slightly alkaline (pH 7.3) conditions for up to 120 min. CZE-UV data showed a continuous degradation of the allergen accompanied by the gradual formation of several reaction products. Characterization of novel allergen variants occurring at longer migration times was done via CZE-ESI-TOF-MS using in-capillary transient capillary isotachophoresis (tCITP) preconcentration to facilitate the identification of minor variants. MS data revealed various modifications of rArt v 3.0201 in response to heating. Variants with deamidations and sulfur-related modifications including both yield and loss of sulfur were identified at increased migration times. Desulfurization produced allergen variants with up to four lanthionines that replaced initial disulfide bonds. In addition, mass spectra revealed shifts in the charge state distribution which indicate concomitant conformational alterations. Moreover, several low-abundant oxidized variants were identified. With extended thermal stress, the portfolio of variants increased and progressively shifted toward rArt v 3.0201 with high lanthionine content. The kinetics of conversion and the complexity of variant composition were pH dependent and increased under alkaline conditions.

Polymer-Enhanced Capillary Transient Isotachophoresis with Boronic Acid-Functionalized Squarylium Dyes for the Fluorescent Determination of Digoxin and Digoxigenin

ABSTRACTHere is reported a new application of polymer-enhanced capillary transient isotachophoresis for the separation and quantification of the drug digoxin and its primary metabolite digoxigenin coupled with laser-induced fluorescence (LIF) detection facilitated by labeling with two boronic acid-functionalized squarylium dyes of different alkyl side chain lengths, SQ-BA1 and SQ-BA2. The conditions for drug–dye complex formation were optimized, as determined by absorbance and fluorescence spectra, according to solution pH and buffer composition. As digoxin has a digitoxose sugar moiety in its structure, it was shown to exhibit better enhancement in the fluorescence intensity of both dyes than digoxigenin, which lacks this moiety, presumably through the formation of a cyclic boronate ester complex. A comparison of analyte labeling in pre-column and on-column modes was conducted in subsequent capillary electrophoresis-LIF studies, with the latter labeling mode yielding superior sensitivity. However, to achieve the complete resolution of labeled digoxin and digoxigenin analytes, it was necessary to use the modified isotachophoresis method, with added borate ions that may differentially interact with the drug and its metabolite, hence affecting their mobilities. Limits of quantification of the method for the determination of digoxin with SQ-BA1 and SQ-BA2 were 2.61 × 10−3 and 2.82 × 10−3 M and limits of detection were 7.83 × 10−4 and 8.47 × 10−4 M while sensitivities were as great as 5.06 × 109 and 2.89 × 109 M−1, respectively, indicating that the method is suitable for practical analysis.

A single-round selection of selective DNA aptamers for mammalian cells by polymer-enhanced capillary transient isotachophoresis.

A single-round DNA aptamer selection for mammalian cells was successfully achieved for the first time using a capillary electrophoresis (CE)-based methodology called polymer-enhanced capillary transient isotachophoresis (PectI). The PectI separation yielded a single peak for the human lung cancer cell line (PC-9) complexed with DNA aptamer candidates, which was effectively separated from a free randomized DNA library peak, ensuring no contamination from free DNA in the PC-9-DNA aptamer complex fraction. The DNA aptamer candidates obtained after a single-round selection employing counter selection with HL-60 were proven to bind selectively and form kinetically stable complexes with PC-9 cells. Interestingly, most aptamer candidates showed high binding ability (Kd = 70-350 nM) with different extents of binding on the cell surface. These facts proved that a single-round selection for mammalian cells by PectI is feasible to obtain various types of aptamer candidates, which have high-affinity even for non-overexpressed but unique targets on the cell surface in addition to overexpressed targets.

Capillary Electrophoresis–Nanoelectrospray Ionization–Selected Reaction Monitoring Mass Spectrometry via a True Sheathless Metal-Coated Emitter Interface for Robust and High-Sensitivity Sample Quantification

A new sheathless transient capillary isotachophoresis (CITP)/capillary zone electrophoresis (CZE)-MS interface, based on a commercially available capillary with an integrated metal-coated ESI emitter, was developed in this study aiming at overcoming the reproducibility and ruggedness problems suffered to a certain degree by almost all the available CE-MS interfaces, and pushing the CE-MS technology suitable for routine sample analysis with high sensitivity. The new CITP/CZE-MS interface allows the electric contact between ESI voltage power supply and the CE separation liquid by using a conductive liquid that comes in contact with the metal-coated surface of the ESI emitter, making it a true sheathless CE-MS interface. Stable electrospray was established by avoiding the formation of gas bubbles from electrochemical reaction inside the CE capillary. Crucial operating parameters, such as sample loading volume, flow rate, and separation voltage, were systematically evaluated for their effects on both CITP/CZE separation efficiency and MS detection sensitivity. Around one hundred CITP/CZE-MS analyses can be easily achieved by using the new sheathless CITP/CZE interface without a noticeable loss of metal coating on the ESI emitter surface, or degrading of the ESI emitter performance. The reproducibility in analyte migration time and quantitative performance of the new interface was experimentally evaluated to demonstrate a LOQ below 5 attomole.

Rapid acquisition of high-affinity DNA aptamer motifs recognizing microbial cell surfaces using polymer-enhanced capillary transient isotachophoresis.

We present a polymer-enhanced capillary transient isotachophoresis (PectI) selection methodology for acquisition of high-affinity (kinetically inert) DNA aptamers capable of recognizing distinct microbial cell surfaces, which requires only a single electrophoretic separation between particles (free cells and cells bound with aptamers) and molecules (unbound or dissociated DNA) in free solution.

Combining capillary electrophoresis and next-generation sequencing for aptamer selection.

Next-generation sequencing (NGS) machines can sequence millions of DNA strands in a single run, such as oligonucleotide (oligo) libraries comprising millions to trillions of discrete oligo sequences. Capillary electrophoresis is an attractive technique to select tight binding oligos or “aptamers” because it requires minimal sample volumes (e.g., 100 nL) and offers a solution-phase selection environment through which enrichment of target-binding oligos can be determined quantitatively. We describe here experiments using capillary transient isotachophoresis (ctITP)-based nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) as a method for selecting aptamers from a randomized library containing a known (29mer) thrombin-binding aptamer. Our capillary electrophoresis (CE)-selected samples were sequenced by the Ion Torrent Personal Genome Machine (PGM) and analyzed for selection efficiency. We show that a single round of CE selection can enrich a randomer synthetic DNA oligo mixture for thrombin-binding activity from 0.4 % aptamer content before selection to >15 % aptamer content.Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-014-8427-y) contains supplementary material, which is available to authorized users.

Facilitating aptamer selection and collection by capillary transient isotachophoresis with laser-induced fluorescence detection

An efficient separation method that utilizes capillary transient isotachophoresis (ctITP) was developed for the preselection of binding ligands. With the ultimate goal of providing enriched fractions from vast libraries for drug discovery, the preselection process described herein entails three distinct elements, which have been validated using a model thrombin protein (target) and thrombin aptamer (ligand) system. First, a high fidelity, on-column labeling scheme employing the noncovalent, fluorescent reagent SYBR Gold was demonstrated for single-stranded DNA with an 11-fold greater sensitivity than pre-column labeling procedures. Second, this on-column labeling was incorporated into a new ctITP method with laser-induced fluorescence (LIF) detection, which provided greatly enhanced resolution of protein-aptamer complex and free aptamer (in comparison to traditional capillary zone electrophoresis (CZE) methods). Third, this enhanced resolution permitted the subsequent accumulation of bound aptamer fractions via an automated collection method, with the establishment of quantitative measures of DNA accumulation. Preselected aptamer or ligand samples such as these can serve as inputs for subsequent lab-on-bead or next-generation-sequencing technologies, enabling accelerated drug discovery.

An Application of Polymer-Enhanced Capillary Transient Isotachophoresis with an Emissive Boronic Acid Functionalized Squarylium Dye as an On-Capillary Labeling Agent for Gram-positive Bacteria

In this paper, the characterization and application of the "PectI" (polymer-enhanced capillary transient isotachophoresis) technique for the separation and detection of same genus, gram-positive bacteria, Bacillus globigii (Bg) and Bacillus subtilis, is demonstrated by employing a boronic acid-functionalized squarylium dye (SQ-BA) as an on-capillary labeling agent, including the quantitative performance and applicability to crude samples. The effect of borate in the separation buffer was also investigated, which revealed that borate strongly affects the separation behavior of bacteria.

On-Column Labeling of Gram-Positive Bacteria with a Boronic Acid Functionalized Squarylium Cyanine Dye for Analysis by Polymer-Enhanced Capillary Transient Isotachophoresis

A new asymmetric, squarylium cyanine dye functionalized by boronic acid ("SQ-BA") was designed and synthesized for on-capillary labeling of gram-positive bacteria to provide for high sensitivity detection by way of a modified form of capillary electrophoresis with laser induced fluorescence detection (CE-LIF). The CE-based separation employed a polymer-enhanced buffer with capillary transient isotachophoresis in a new hybrid method dubbed "PectI." It was found that the addition of various monosaccharides to SQ-BA in a batch aqueous solution greatly enhanced the emission of the boronic acid functionalized dye by a factor of up to 18.3 at a long wavelength (λ(ex) = 630 nm, λ(em) = 660 nm) with a high affinity constant (K = ~10(2.80) M(-1)) superior to other sugar probes. Semiempirical quantum mechanics calculations suggest that the mechanism for this high enhancement may involve the dissociation of initially nonemissive dye associates (stabilized by an intramolecular hydrogen bond) upon complex formation with sugars. The fluorescence emission of SQ-BA was also significantly enhanced in the presence of a gram-positive bacterial spore, Bacillus globigii (Bg), which serves as a simulant of B. anthracis (or anthrax) and which possesses a peptidoglycan (sugar)-rich spore coat to provide ample sites for interaction with the dye. Several peaks were observed for a pure Bg sample even with polyethyleneoxide (PEO) present in the CE separation buffer, despite the polymer's previously demonstrated ability to focus microoorganisms to a single peak during migration. Likewise, several peaks were observed for a Bg sample when capillary transient isotachophoresis (ctITP) alone was employed. However, the new combination of these techniques as "PectI" dramatically and reproducibly focused the bacteria to a single peak with no staining procedure. Using PectI, the trace detection of Bg spores (corresponding to approximately three cells per injection) along with separation efficiency enough to separate Bg from another gram-positive bacteria, Saccharomyces cerevisiae (resolution, R(s) = 6.09, and apparent plate number, N = 2.7-3.3 × 10(5)), were successfully achieved.

Advances in the analysis of proteins and peptides by capillary electrophoresis with matrix‐assisted laser desorption/ionization and electrospray‐mass spectrometry detection

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.