Coupling Of Capillary Electrophoresis Research Articles

Highly sensitive two-dimensional profiling of N-linked glycans by hydrophilic interaction liquid chromatography and dual stacking capillary gel electrophoresis

BackgroundN-Glycosylation is one of the most important post-translational modifications in proteins. As the N-glycan profiles in biological samples are diverse and change according to the pathological condition, various profiling methods have been developed, such as liquid chromatography (LC), capillary electrophoresis (CE), and mass spectrometry. However, conventional analytical methods have limitations in sensitivity and/or resolution, hindering the discovery of minor but specific N-glycans that are important both in the basic glycobiology research and in the medical application as biomarkers. Therefore, a highly sensitive and high-resolution N-glycan profiling method is required. ResultsIn this study, we developed a novel two-dimensional (2D) separation system, which couples hydrophilic interaction liquid chromatography (HILIC) with capillary gel electrophoresis (CGE) via large-volume dual preconcentration by isotachophoresis and stacking (LDIS). Owing to the efficient preconcentration efficiency of LDIS, limit of detection reached 12 pM (60 amol, S/N = 3) with good calibration curve linearity (R2 > 0.999) in the 2D analysis of maltoheptaose. Finally, 2D profiling of N-glycans obtained from standard glycoproteins and cell lysates were demonstrated. High-resolution 2D profiles were successfully obtained by data alignment using triple internal standards. N-glycans were well distributed on the HILIC/CGE 2D plane based on the glycan size, number of sialic acids, linkage type, and so on. As a result, specific minor glycans were successfully identified in HepG2 and HeLa cell lysates. Significance and noveltyIn conclusion, the HILIC/CGE 2D analysis method showed sufficient sensitivity and resolution for identifying minor but specific N-glycans from complicated cellular samples, indicating the potential as a next-generation N-glycomics tool. Our novel approach for coupling LC and CE can also dramatically improve the sensitivity in other separation modes, which can be a new standard of 2D bioanalysis applicable not only to glycans, but also to other diverse biomolecules such as metabolites, proteins, and nucleic acids.

Comparative study of the vibrating capillary nebulizer (VCN) and commercially available interfaces for on-line coupling of capillary electrophoresis with ICP-MS.

Capillary electrophoresis (CE) is a powerful and sensitive tool for speciation analysis when combined with inductively coupled plasmamass spectrometry (ICP-MS); however, the performance of this technique can be limited by the nature of pneumatic nebulizers. This study compares two commercially available pneumatic nebulizers to a newly introduced vibrating capillary nebulizer (VCN) for on-line coupling of CE with ICP-MS. The VCN is a low-cost, non-pneumatic nebulizer that is based on the design of capillary vibrating sharp-edge spray ionization. As a piezoelectrically driven nebulization source, the VCN creates an aerosol independent of gas flows and does not produce a low-pressure region at the nebulizer orifice. To compare the systems, we performed replicate analyses of sulfate in river water with each nebulizer and the same CE and ICP-MS instruments and determined the figures of merit of each setup. With the CE-VCN-ICP-MS setup, we achieved around 2-4 times lower sensitivity compared to the commercial setups. However, the VCN-based setup provided lower noise levels and better linear correlation from the analysis of calibration standards, which resulted in indistinguishable LOD and LOQ values from the in-house-built VCN-based and commercial setups for CE-ICP-MS analysis. The VCN is found to have the highest baseline stability with a standard deviation of 3500 cts s-1, corresponding to an RSD of 2.7%. High reproducibility is found with the VCN with a peak area RSD of 4.1% between 3 replicate measurements.

Application of capillary electrophoresis with capacitively contactless conductivity detection for biomedical analysis.

The coupling of capillary electrophoresis (CE) with capacitively coupled contactless conductivity detection (C4 D) has become convenient analytical method for determination of small molecules that do not possess chromogenic or fluorogenic group. The implementations of CE with C4 D in the determination of inorganic and organic ions and amino acids in biomedical field are demonstrated. Attention on background electrolyte composition, sample treatment procedures, and the utilize of multi-detection systems are described. A number of tables summarizing highly developed CE-C4 D methods and the figures of merit attained are involved. Lastly, concluding remarks and perspectives are argued.

Coupling capillary electrophoresis with mass spectrometry for the analysis of oxidized phospholipids in human high-density lipoproteins.

The oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) products in human high-density lipoproteins (HDLs) were investigated by low-flow capillary electrophoresis-mass spectrometry (low-flow CE-MS). To accelerate the optimization, native PAPC (n-PAPC) standard was first analyzed by a commercial CE instrument with a photodiode array detector. The optimal separation buffer contained 60% (v/v) acetonitrile, 40% (v/v) methanol, 20mM ammonium acetate, 0.5% (v/v) formic acid, and 0.1% (v/v) water. The selected separation voltage and capillary temperature were 20kV and 23°C. The optimal CE separation buffer was then used for the low-flow CE-MS analysis. The selected MS conditions contained heated capillary temperature (250°C), capillary voltage (10V), and injection time (1 s). No sheath gas was used for MS. The linear range for n-PAPC was 2.5-100.0µg/mL. The coefficient of determination (R2 ) was 0.9918. The concentration limit of detection was 1.52µg/mL, and the concentration limit of quantitation was 4.60µg/mL. The optimal low-flow CE-MS method showed good repeatability and sensitivity. The ox-PAPC products in human HDLs were determined based on the in vitro ox-PAPC products of n-PAPC standard. Twenty-one ox-PAPC products have been analyzed in human HDLs. Uremic patients showed significantly higher levels of 15 ox-PAPC products than healthy subjects.

Capillary zone electrophoresis-high field asymmetric ion mobility spectrometry-tandem mass spectrometry for top-down characterization of histone proteoforms.

Characterization of histone proteoforms with various post-translational modifications (PTMs) is critical for a better understanding of functions of histone proteoforms in epigenetic control of gene expression. Mass spectrometry (MS)-based top-down proteomics (TDP) is a valuable approach for delineating histone proteoforms because it can provide us with a bird's-eye view of histone proteoforms carrying diverse combinations of PTMs. Here, we present the first example of coupling capillary zone electrophoresis (CZE), ion mobility spectrometry (IMS), and MS for online multi-dimensional separations of histone proteoforms. Our CZE-high-field asymmetric waveform IMS (FAIMS)-MS/MS platform identified 366 (ProSight PD) and 602 (TopPIC) histone proteoforms from a commercial calf histone sample using a low microgram amount of histone sample as the starting material. CZE-FAIMS-MS/MS improved the number of histone proteoform identifications by about 3 folds compared to CZE-MS/MS alone (without FAIMS). The results indicate that CZE-FAIMS-MS/MS could be a useful tool for comprehensive characterization of histone proteoforms with high sensitivity.

Open Port Interface for Coupling Capillary Electrophoresis and Mass Spectrometry: Performance Evaluation for Capillary Isoelectric Focusing.

Capillary electrophoresis (CE) combined with mass spectrometry (MS) is a powerful analytical technique that utilizes the resolving power of CE and the mass-detection capabilities of MS. In many cases, CE is coupled to MS via a sheath-flow interface (SFI). This interface has a simple design and can be easily constructed; however, it often suffers from issues such as MS signal suppression, interference of MS and CE electrical circuits, and the inability to set an optical point of detection close to the capillary end due to the specific design of the coupling union. In this paper, we describe a novel coupling of CE and MS based upon the open port interface (OPI). The OPI differs from classical sheath flow interfaces by operating at flow rates at least 1 order of magnitude higher. In addition to the flow rate difference, the OPI provides more efficient mixing of the capillary eluates with the transport fluid and thus minimizes MS signal suppression. In this work, we compared the performance of OPI and SFI in a series of capillary isoelectric focusing (cIEF) experiments with 5 pI markers, carbonic anhydrase II and NIST antibody. The evaluation criteria for the comparison of the OPI and SFI were analytical sensitivity, reproducibility, and pI marker linearity. Given the extent of sample dilution in the OPI, we also compared the peak resolution determined using an upstream UV detector to those determined by the downstream mass spectrometer. The results suggested that the OPI configuration reduced signal suppression, with no adverse effect on peak resolution. In addition, the OPI provided better decoupling of the CE and MS potentials as well as reduced signal dependence upon the sheath liquid composition. While these results are preliminary, they suggest that the OPI is a viable approach for CE-MS coupling.

Submersible Capillary Electrophoresis Analyzer: A Proof-of-Concept Demonstration of an In Situ Instrument for Future Missions to Ocean Worlds

We report here the first fully automated capillary electrophoresis (CE) system that can be operated underwater. The system performs sample acquisition and analysis by coupling CE to contactless conductivity detection. Using 5 M acetic acid as the background electrolyte (BGE), inorganic cations and amino acids at concentrations as low as 5.2 μM can be separated and identified. This technology could be augmented to include a variety of other detection modes. This system serves as an early prototype for potential future underwater explorers on ocean worlds of the outer solar system such as Europa or Enceladus. This work documents the first step in the development of this general-purpose technology platform.

Coupling capillary electrophoresis-electrospray ionization mass spectrometry with sheathless interface for drug analysis

Capillary electrophoresis-mass spectrometry （CE-MS） combines the advantages of capillary electrophoresis， such as the high separation efficiency and low sample consumption， and the high detection sensitivity of mass spectrometry and the ability for providing the structural information for structure elucidation of unknown components. However， the interface technology for coupling capillary electrophoresis and mass spectrometry is still not well resolved. In the present work， we explored the application of the sheathless CE-MS interface which was prepared by gold foil-wrapped CE separation column tip directly as a spray electrode for the analysis of five tyrosine kinase inhibitors， namely sunitinib， imatinib mesylate， gefitinib， dasatinib and erlotinib. This interface integrates separation and electrospray ionization in one capillary， which is easy to manufacture， low in cost， and can be produced in batches. We found that using the nonaqueous CE separation mode can not only achieve baseline separation of five tyrosine kinase inhibitors， but also obtain stable mass spectrometry signals. First， we investigated the effect of the electrolyte solution composition on the separation. The optimized background electrolyte composition was obtained： 2% （v/v） acetic acid and 5 mmol/L ammonium acetate in acetonitrile-methanol （80∶20， v/v）. Under optimized conditions， the five kinase inhibitors could be baseline separated， meantime， the sheathless interface could also maintain stable electrospray for a long time. The relative standard deviation （RSD） values of the intraday and interday repeatability of the analyte retention times were less than 0.5% and 0.8%， respectively， and the RSD value between interface batches is less than 2.6%. Compared with CE-MS with aqueous phase， the separation column efficiency of the five tyrosine kinase inhibitors under nonaqueous phase conditions is higher， the detection sensitivity is higher， and the absolute detection limit reaches amol level. In addition， we evaluated the sheathless interface with various organic acids， such as palaflin A， salvianolic acid C， and rosmarinic acid， as well as hydrophobic macrolide antibiotics， azithromycin， erythromycin， and sporin A， good separation effect and mass spectrometric detection results can be obtained.

Offline Coupling of Asymmetrical Flow Field-Flow Fractionation and Capillary Electrophoresis for Separation of Extracellular Vesicles.

Extracellular vesicles (EVs) play important roles in cell-to-cell communications and carry high potential as markers targeted in disease diagnosis, prognosis, and therapeutic development. The main obstacles to EV study are their high heterogeneity; low amounts present in samples; and physical similarity to the abundant, interfering matrix components. Multiple rounds of separation and purification are often needed prior to EV characterization and function assessment. Herein, we report the offline coupling of asymmetrical flow field-flow fractionation (AF4) and capillary electrophoresis (CE) for EV analysis. While AF4 provides gentle and fast EV separation by size, CE resolves EVs from contaminants with similar sizes but different surface charges. Employing Western Blotting, ELISA, and SEM, we confirmed that intact EVs were eluted within a stable time window under the optimal AF4 and CE conditions. We also proved that EVs could be resolved from free proteins and high-density lipoproteins by AF4 and be further separated from the low-density lipoproteins co-eluted in AF4. The effectiveness of the coupled AF4-CE system in EV analysis was demonstrated by monitoring the changes in EV secretion from cells and by direct injection of human serum and detection of serum EVs. We believe that coupling AF4 and CE can provide rapid EV quantification in biological samples with much reduced matrix interference and be valuable for the study of total EVs and EV subpopulations produced by cells or present in clinical samples.

Nanoflow Sheath Voltage-Free Interfacing of Capillary Electrophoresis and Mass Spectrometry for the Detection of Small Molecules.

Coupling capillary electrophoresis (CE) to mass spectrometry (MS) is a powerful strategy to leverage a high separation efficiency with structural identification. Traditional CE-MS interfacing relies upon voltage to drive this process. Additionally, sheathless interfacing requires that the electrophoresis generates a sufficient volumetric flow to sustain the ionization process. Vibrating sharp-edge spray ionization (VSSI) is a new method to interface capillary electrophoresis to mass analyzers. In contrast to traditional interfacing, VSSI is voltage-free, making it straightforward for CE and MS. New nanoflow sheath CE-VSSI-MS is introduced in this work to reduce the reliance on the separation flow rate to facilitate the transfer of analyte to the MS. The nanoflow sheath VSSI spray ionization functions from 400 to 900 nL/min. Using the new nanoflow sheath reported here, volumetric flow rate through the separation capillary is less critical, allowing the use of a small (i.e., 20 to 25 μm) inner diameter separation capillary and enabling the use of higher separation voltages and faster analysis. Moreover, the use of a nanoflow sheath enables greater flexibility in the separation conditions. The nanoflow sheath is operated using aqueous solutions in the background electrolyte and in the sheath, demonstrating the separation can be performed under normal and reversed polarity in the presence or absence of electroosmotic flow. This includes the use of a wider pH range as well. The versatility of nanoflow sheath CE-VSSI-MS is demonstrated by separating cationic, anionic, and zwitterionic molecules under a variety of separation conditions. The detection sensitivity observed with nanoflow sheath CE-VSSI-MS is comparable to that obtained with sheathless CE-VSSI-MS as well as CE-MS separations with electrospray ionization interfacing. A bare fused silica capillary is used to separate cationic β-blockers with a near-neutral background electrolyte at concentrations ranging from 1.0 nM to 1.0 μM. Under acidic conditions, 13 amino acids are separated with normal polarity at a concentration ranging from 0.25 to 5 μM. Finally, separations of anionic compounds are demonstrated using reversed polarity under conditions of suppressed electroosmotic flow through the use of a semipermanent surface coating. With a near-neutral separation electrolyte, anionic nonsteroidal anti-inflammatory drugs are detected over a concentration range of 0.1 to 5.0 μM.

Microfabricated Liquid Junction Capillary Electrophoresis-Mass Spectrometry Interface.

Coupling of capillary electrophoresis (CE) with mass spectrometry (MS) represents a powerful combination for performing rapid, efficient, and sensitive analysis of a variety of compounds. Here we describe a construction, operation, and application of a microfabricated liquid junction CE-MS interface. The interface is designed as a microfabricated unit with an integrated liquid junction and electrospray tip made from polyimide, which is positioned in a plastic connection block securing the separation CE capillary and attachable to the CE instrument. The application was demonstrated by CE-MS analysis of dextran oligomers labeled by (2-aminoethyl)trimethylammonium (AETMA) salt.

Simultaneous total and speciation analysis of rhenium by capillary electrophoresis-inductively coupled plasma mass spectrometry

As one of the most concerned radionuclides, 99technetium (Tc) usually exists as pertechnetate (TcO4−) and low-valence Tc(IV) in the environment. The speciation analysis of 99Tc plays a crucial role in understanding its toxicity, bioavailability and mobility, and developing effective remediation strategies. However, conventional hyphenated techniques for 99Tc speciation analysis usually require a large sample volume and tedious operation process. In this work, a simple but highly effective system, coupling capillary electrophoresis to inductively coupled plasma mass spectrometry (CE-ICP-MS), was developed to analyze total rhenium (TRe), Re(IV), and Re(VII), known as the analogues of technetium. It is worth noting that by the use of a sprayer with a direct-injection nebulizer as the interface, the CE and ICP-MS were efficiently coupled as an entity that shares the advantages of both modules. In comparison to conventional techniques, the proposed CE-ICP-MS dominates in terms of less sample consumption and higher sample introduction efficiency, thus results in improved sensitivity, reduced radioactive waste, and more significantly, alleviated radiological exposure to operators. Under the optimized conditions, LODs of 0.02 μg L−1 and 0.01 μg L−1 were obtained for TRe and Re(VII), respectively, with relative standard deviations (RSDs) better than 3.0%. In addition, the accuracy and practicability of this hyphenated system were validated by analyzing simulated Hanford site groundwater samples with satisfactory recovery and separation of TRe, Re(IV), and Re(VII). The proposed method retains the great potential for the speciation analysis of 99Tc in various samples.

Intracellular silver speciation by coupling capillary electrophoresis to ICP-MS integrating a high performance spiral flow spray chamber

The study of silver species and their distribution/transformation in cell interior is of high significance for the elucidation of toxicology of silver nanoparticles (AgNPs). The intracellular speciation of dissolved Ag(I) and AgNPs was reported. The analytical platform integrated capillary electrophoresis (CE) to inductively coupled plasma-mass spectrometry (ICP-MS) incorporating a high efficiency interface and a high performance spiral flow spray chamber (SFSC). The interface and the SFSC provide a favorable detection limit of 87 ng L−1 for the dissolved Ag(I). Total silver content was quantified by ICP-MS subject to digestion of the cell lysate, and quantification of AgNPs was carried out by subtraction. The speciation of dissolved Ag(I) and AgNPs in culture medium and HepG2 cells was performed, with RSDs of <3% for relative peak area and <2% for migration time, as well as spiking recoveries of 93.8%–94.3% in opti-MEM and 92.7%–106.6% in cell lysate. The present study indicated higher solubility of AgNPs in the cell interior with respect to that in the culture medium, due to oxidative stress or acidic microenvironment in the cancer cells.

Indirect capillary electrophoresis immunoassay of membrane protein in extracellular vesicles.

Extracellular vesicles (EVs) exist in biological fluids such as blood, urine, and cerebrospinal fluid, and these have shown promise for use as biomarkers of cancers. Conventional methods for determination of EVs include direct detection via enzyme-linked immunosorbent assay and detection of their membrane proteins via western blotting. These techniques, however, have individual shortcomings in terms of the need for large sample consumption, processes that are time-consuming, and a lack of the capacity for quantification. In this study, we developed a method to determine the EV membrane protein, CD63, by coupling capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF). In this process, the EVs were isolated from a culture medium and were subsequently reacted with a fluorescently labeled anti-CD63 antibody to form a CD63 complex localized on the surface of EVs. After removing the EVs containing the CD63 immune complex by centrifugation, the supernatant containing the free fluorescent antibody was injected into a capillary to serve as a sample. A decrease in the peak area of the free fluorescent antibody became apparent when the amount of EVs was increased while that of the fluorescent antibody remained constant. The peak areas were decreased proportionally against the increased amounts of EVs. The concentration of the CD63 could then be estimated based on the slope of the linear relationship. This study is the first to quantify CD63 immobilized on EVs via CEIA-LIF, which is a novel method with the potential to determine membrane proteins localized on the surface of EVs.

A gold foil covered fused silica capillary tip as a sheathless interface for coupling capillary electrophoresis-mass spectrometry

A method for the preparation of an on-column ESI emitter used as the sheathless interface for coupling capillary electrophoresis (CE) with mass spectrometry (MS) was developed. The emitter was directly fabricated at the outlet end of the separation capillary which was etched with HF solution to a symmetrical tip. The tip was covered with a small piece of gold foil which was fixed by epoxy resin glue for electrical contact. Such a prepared ESI emitter can produce a stable ESI signal over the wide range of flow rate from 50 nL/min to 800 nL/min. The performance of the CE-MS with the sheathless interface was evaluated by using the separation of four alkaloids. It was found that the strong electroosmotic flow produced by the multiple polyelectrolyte coating on the capillary is necessary for maintaining a stable MS signal. Effect of the running buffer composition, concentration and the CE separation voltages on the ESI signal strength were investigated. The absolute detection limits for the alkaloids was determined as fmol level. Moreover, the CE-MS was applied for the analyses of trypsin digestion of cytochrome C and small molecular organic anions. The emitter performed very stable with a lifetime of at least 180 h.

On-line coupling of capillary electrophoresis with microdialysis for determining saccharides in dairy products and honey

Free sucrose, lactose, galactose, glucose and fructose were determined in yoghurts, milk and honey using on-line coupling of capillary electrophoresis with microdialysis. The dairy products were diluted 50-fold with 10 mmol/L NaOH and sampled using laboratory-made microdialysis probes. The microdialysate was brought to the entrance of the electrophoretic capillary and the coupling consisted in a polydimethylsiloxane (PDMS) cross connector working in the flow-gating interface regime. The electrophoretic analysis was performed in 50 mmol/L NaOH (pH 12.6) background electrolyte, where baseline separation of the five saccharides was achieved in 3.5 min. The LOQs varied in the range 2.3–7.3 mg/L, the number of separation plates varied between 176,000 plates/m for glucose to 326,000 plates/m for galactose and the relative standard deviation (RSD) for ten consecutive analyses of fruit yoghurt was 0.2% for the migration time and 4.4–7.6% for the peak area.

The NpV and PuV Carbonate Systems: Thermodynamics and Coordination Chemistry

New stability constants for NpV and PuV carbonates have been established by coupling capillary electrophoresis (CE) and ICP‐MS at the ionic strength of 0.202 mol/kgwater in NaClO4 and at various temperatures. The thermodynamic data [variation of the molar enthalpy, ΔrHm(T°) and entropy, ΔrSm(T) of reaction] associated with the formation of the AnO2CO3– (An = Np, Pu), AnO2(CO3)23–, and AnO2(CO4)35– complexes were determined. A drastic decrease in molar entropy was observed between the second and third complexes, suggesting important structural changes. Ab initio molecular dynamics showed that the 1:3 complex is formed with two carbonate ligands located at a shorter distance and one at a longer distance, suggesting that the closer ligands are coordinated in a bidentate fashion to the plutonyl(V) moiety whereas the third ligand is monodentate. The variation of the electrophoretic mobilities as function of charge/size ratio confirms unambiguously that the triscarbonate complex of pentavalent plutonium structurally differs from that of hexavalent plutonium where all carbonates anions are bidentate.

Capillary electrophoresis-based approaches for the study of affinity interactions combined with various sensitive and nontraditional detection techniques.

Nearly all processes in living organisms are controlled and regulated by the synergy of many biomolecule interactions involving proteins, peptides, nucleic acids, nucleotides, saccharides, and small molecular weight ligands. There is growing interest in understanding them, not only for the purposes of interactomics as an essential part of system biology, but also in their further elucidation in disease pathology, diagnostics, and treatment. The necessity of detailed investigation of these interactions leads to the requirement of laboratory methods characterized by high efficiency and sensitivity. As a result, many instrumental approaches differing in their fundamental principles have been developed, including those based on capillary electrophoresis. Although capillary electrophoresis offers numerous advantages for such studies, it still has one serious limitation, its poor concentration sensitivity with the most commonly used detection method-ultraviolet-visible spectrometry. However, coupling capillary electrophoresis with a more sensitive detector fulfils the above-mentioned requirement. In this review, capillary electrophoresis combined with fluorescence, mass spectrometry, and several nontraditional detection techniques in affinity interaction studies are summarized and discussed, together with the possibility of conducting these measurements in microchip format.

Recent Advances and New Perspectives in Capillary Electrophoresis-Mass Spectrometry for Single Cell "Omics".

Accurate clinical therapeutics rely on understanding the metabolic responses of individual cells. However, the high level of heterogeneity between cells means that simply sampling from large populations of cells is not necessarily a reliable approximation of an individual cell’s response. As a result, there have been numerous developments in the field of single-cell analysis to address this lack of knowledge. Many of these developments have focused on the coupling of capillary electrophoresis (CE), a separation technique with low sample consumption and high resolving power, and mass spectrometry (MS), a sensitive detection method for interrogating all ions in a sample in a single analysis. In recent years, there have been many notable advancements at each step of the single-cell CE-MS analysis workflow, including sampling, manipulation, separation, and MS analysis. In each of these areas, the combined improvements in analytical instrumentation and achievements of numerous researchers have served to drive the field forward to new frontiers. Consequently, notable biological discoveries have been made possible by the implementation of these methods. Although there is still room in the field for numerous further advances, researchers have effectively minimized various limitations in detection of analytes, and it is expected that there will be many more developments in the near future.

Profiling of N-linked glycans from 100 cells by capillary electrophoresis with large-volume dual preconcentration by isotachophoresis and stacking

Glycan structure is changed in response with pathogenesis like cancer. Profiling of glycans from limited number of pathogenetic cells in an early-stage tissue is essential for discovering effective drugs. For analyzing tiny biological samples, we developed sensitive, high-resolution, and salt-tolerant method for analyzing trace level of N-linked glycans by coupling capillary electrophoresis (CE), laser-induced fluorescence (LIF) detection, and a new online sample preconcentration (OSP) method named “large-volume dual preconcentration by isotachophoresis and stacking (LDIS)”, which is composed of two OSP methods, large-volume sample stacking (LVSS) and transient isotachophoresis (tITP). A typical LDIS-CE-LIF protocol was simple: a short-plug of leading electrolyte (LE) and large-volume sample solution were introduced to a capillary, followed by application of constant voltage. In the analysis of glucose ladder labeled with 8-aminopyrene-1,3,6-trisulfonic acid with 10 mM sodium chloride as LE, up to 2300-fold sensitivity increase was achieved with higher resolution than those in normal CE. By applying pressure assist during preconcentration, both viscous gel electrolyte and salty matrix of up to 10 mM NaCl were acceptable. Finally, N-glycans from approximately 100 cells (HeLa, MCF7, and HepG2) were analyzed as the model of localized tumor cells. From 30 to 40 glycans were successfully detected with almost same profile of large-scale sample. N-glycan structure could be predicted by searching glucose-unit value via Glycobase database, indicating that HepG2 expressed more sialylated glycans and MCF-7 expressed less glycans respectively, comparing with HeLa cells. It suggests the potential of LDIS-CE-LIF for discovery of disease-specific N-linked glycans in microscale environment.