Capillary Zone Electrophoresis of 8-Aminopyrene-1,3,6-trisulfonic Acid Labeled Carbohydrates with Online Electrokinetic Sample Cleanup.

Capillary electrophoresis is one of the frequently used separation techniques for the analysis of complex carbohydrates. Since sugars lack chromophore or fluorophore groups, their capillary electrophoresis analysis usually requires tagging by a charged fluorophore. To speed up the derivatization reaction, a large excess of the labeling reagent is typically used; therefore, a purification step is necessary prior to CE analysis using the industry standard low-pH gel-buffer system. In addition to representing an extra sample preparation step with the associated labor and cost, the purification process also holds the risk of losing some of the sample components. In this paper we introduce an online electrokinetic sample cleanup process with electroosmotic flow (EOF)-assisted separation in a bare fused silica capillary using alkaline pH background electrolyte and normal polarity separation voltage. 8-Aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled maltooligosaccharides were analyzed first to understand the complex effect of the downstream EOF and the counter current electromigration of the sample components including the labeling dye. The use of 150 mM caproic acid-253 mM Tris (pH 8.1) running buffer facilitated the entrance of the sample components of interest into the separation capillary, while the excess labeling reagent was excluded and, therefore, did not interfere with the detection. The alkaline caproic acid-Tris running buffer was then applied to the N-glycome analysis of human serum samples, showing excellent separation performance, and more importantly, the extra sample purification step was not required.

Microbead-based extracorporeal immuno-affinity virus capture: a feasibility study to address the SARS-CoV-2 pandemic.

In this paper, we report on the utilization of micro-technology based tools to fight viral infections. Inspired by various hemoperfusion and immune-affinity capture systems, a blood virus depletion device has been developed that offers highly efficient capture and removal of the targeted virus from the circulation, thus decreasing virus load. Single-domain antibodies against the Wuhan (VHH-72) virus strain produced by recombinant DNA technology were immobilized on the surface of glass micro-beads, which were then utilized as stationary phase. For feasibility testing, the virus suspension was flown through the prototype immune-affinity device that captured the viruses and the filtered media left the column. The feasibility test of the proposed technology was performed in a Biosafety Level 4 classified laboratory using the Wuhan SARS-CoV-2 strain. The laboratory scale device actually captured 120,000 virus particles from the culture media circulation proving the feasibility of the suggested technology. This performance has an estimated capture ability of 15 million virus particles by using the therapeutic size column design, representing three times over-engineering with the assumption of 5 million genomic virus copies in an average viremic patient. Our results suggested that this new therapeutic virus capture device could significantly lower virus load thus preventing the development of more severe COVID-19 cases and consequently reducing mortality rate.

The Effect of Sample Glucose Content on PNGase F-Mediated N-Glycan Release Analyzed by Capillary Electrophoresis.

Protein therapeutics have recently gained high importance in general health care along with applied clinical research. Therefore, it is important to understand the structure-function relationship of these new generation drugs. Asparagine-bound carbohydrates represent an important critical quality attribute of therapeutic glycoproteins, reportedly impacting the efficacy, immunogenicity, clearance rate, stability, solubility, pharmacokinetics and mode of action of the product. In most instances, these linked N-glycans are analyzed in their unconjugated form after endoglycosidase-mediated release, e.g., PNGase F-mediated liberation. In this paper, first, N-glycan release kinetics were evaluated using our previously reported in-house produced 6His-PNGase F enzyme. The resulting deglycosylation products were quantified by sodium dodecyl sulfate capillary gel electrophoresis to determine the optimal digestion time. Next, the effect of sample glucose content was investigated as a potential endoglycosidase activity modifier. A comparative Michaelis-Menten kinetics study was performed between the 6His-PNGase F and a frequently employed commercial PNGase F product with and without the presence of glucose in the digestion reaction mixture. It was found that 1 mg/mL glucose in the sample activated the 6His-PNGase F enzyme, while did not affect the release efficiency of the commercial PNGase F. Capillary isoelectric focusing revealed subtle charge heterogeneity differences between the two endoglycosidases, manifested by the lack of extra acidic charge variants in the cIEF trace of the 6His-PNGase F enzyme, which might have possibly influenced the glucose-mediated enzyme activity differences.

Time-Resolved SAXS Study of Polarity- and Surfactant-Controlled Superlattice Transformations of Oleate-Capped Nanocubes During Solvent Removal.

Structural transformations and lattice expansion of oleate-capped iron oxide nanocube superlattices are studied by time-resolved small-angle X-ray scattering (SAXS) during solvent removal. The combination of conductor-like screening model for real solvents (COSMO-RS) theory with computational fluid dynamics (CFD) modeling provides information on the solvent composition and polarity during droplet evaporation. Evaporation-driven poor-solvent enrichment in the presence of free oleic acid results in the formation of superlattices with a tilted face-centered cubic (fcc) structure when the polarity reaches its maximum. The tilted fcc lattice expands subsequently during the removal of the poor solvent and eventually transforms to a regular simple cubic (sc) lattice during the final evaporation stage when only free oleic acid remains. Comparative studies show that both the increase in polarity as the poor solvent is enriched and the presence of a sufficient amount of added oleic acid is required to promote the formation of structurally diverse superlattices with large domain sizes.

N-Glycosylation Profiling of Human Blood in Type 2 Diabetes by Capillary Electrophoresis: A Preliminary Study.

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis bioanalytical technique, for a high-resolution determination of the N-glycan profile in human blood samples of patients with type 2 diabetes (T2D). To achieve the profile information of these complex oligosaccharides, linked by asparagine to hIgG in the blood, the glycoproteins of the samples needed to be cleaved, labelled, and purified with sufficient yield and selectivity. The resulting samples were analyzed by capillary electrophoresis, with laser-induced fluorescence detection. After separation parameter optimization, the capillary electrophoresis technique was implemented for efficient N-glycan profiling of whole blood samples from the diabetic patients. Our results revealed that there were subtle differences between the N-glycan profiles of the diabetic and control samples; in particular, two N-glycan structures were identified as potential glycobiomarkers that could reveal significant changes between the untreated/treated type 2 diabetic and control samples. By analyzing the resulting oligosaccharide profiles, clinically relevant information was obtained, revealing the differences between the untreated and HMG-CoA reductase-inhibitor-treated diabetic patients on changes in the N-glycan profile in the blood. In addition, the information from specific IgG N-glycosylation profiles in T2D could shed light on underlying inflammatory pathophysiological processes and lead to drug targets.

Integrated workflow for urinary prostate specific antigen N-glycosylation analysis using sdAb partitioning and downstream capillary electrophoresis separation.

Prostate cancer represents the second highest malignancy rate in men in all cancer diagnoses worldwide. The development and progression of prostate cancer is not completely understood yet at molecular level, but it has been reported that changes in the N-glycosylation of prostate-specific antigen (PSA) occur during tumor genesis. In this paper we report on the development and implementation of a high-throughput capillary electrophoresis based glycan analysis workflow for urinary PSA analysis. The technology utilizes selective, high yield single domain antibody based PSA capture, followed by preconcentration and capillary electrophoresis coupled with laser-induced fluorescence detection, resulting in high resolution N-glycan profiles. Urinary PSA glycan profiles were compared to a commercially available PSA standard revealing differences in their α2,3- and α2,6-sialylated isomers, proving the excellent selectivity of the suggested workflow. This is important as sialylation classification plays an important role in the differentiation between indolent, significant and aggressive forms of prostate cancer.

Modification of Hemodialysis Membranes for Efficient Circulating Tumor Cell Capture for Cancer Therapy.

BACKGROUND: It is well known that more than 90% of cancer deaths are due to metastases. However, the entire tumorigenesis process is not fully understood, and it is evident that cells spreading from the primary tumor play a key role in initiating the metastatic process. Tumor proliferation and invasion also elevate the concentration of regular and irregular metabolites in the serum, which may alter the normal function of the entire human homeostasis and possibly causes cancer metabolism syndrome, also referred to as cachexia. METHODS: We report on the modification of commercially available hemodialysis membranes to selectively capture circulating tumor cells from the blood stream by means of immobilized human anti-EpCAM antibodies on the inner surface of the fibers. All critical steps are described that required in situ addition of the immuno-affinity feature to hemodialyzer cartridges in order to capture EpCAM positive circulating tumor cells, which represents ~80% of cancer cell types. RESULTS: The cell capture efficiency of the suggested technology was demonstrated by spiking HCT116 cancer cells both into buffer solution and whole blood and run through on the modified cartridge. Flow cytometry was used to quantitatively evaluate the cell clearance performance of the approach. CONCLUSIONS: The suggested modification has no significant effect on the porous structure of the hemodialysis membranes; it keeps its cytokine removal capability, addressing cachexia simultaneously with CTC removal.

Recent advances in the analysis of human milk oligosaccharides by liquid phase separation methods.

Human milk is a complex, dynamically changing biological fluid, which contains a large amount of non-conjugated carbohydrates, referred to as human milk oligosaccharides (HMOs). These HMOs are very important for the infants as they play important roles in the formation of the gut microbiome, the immune system and support brain development. HMOs show highly complex structural diversity due to numerous linkage possibilities of the building monosaccharides. In order to elucidate their structure-function relationship and to develop more effective infant formulas, cutting-edge analytical technologies are in great demand. In this paper, we review the current strategies for HMO analysis based on liquid phase separation methods. High performance liquid chromatography, capillary electrophoresis and their hyphenation with mass spectrometry are critically reviewed, emphasizing their advantages and disadvantages from practical point of views. Recent advances of the methods are categorized according to their application fields.

Separation based characterization methods for the N-glycosylation analysis of prostate-specific antigen.

Prostate cancer has the highest malignancy rate diagnosed in men worldwide. Albeit, the gold standard serum prostate-specific antigen (PSA) assays reduced the mortality rate of the disease, the number of false positive diagnoses steeply increased. Therefore, there is an urgent need for complementary biomarkers to enhance the specificity and selectivity of current diagnostic methods. Information about PSA glycosylation can help to fulfill this gap as alterations of its carbohydrate moieties due to cancerous transformation may represent additional markers to distinguish malignant from benign tumors. However, development of suitable methods and instrumentations to investigate the N-glycosylation profile of PSA represents a challenge. In this paper, we critically review the current bioanalytical trends and strategies in the field of PSA glycobiomarker research focusing on separation based characterization methods.

Evaluation of Possible Processing Time Effects on the Global N-Glycosylation Profile of Human Blood Samples.

The utilization of N-glycan profiling recently gained high importance in fundamental biomedical and applied clinical research. However, for the time being, no glycan biomarker has been approved for clinical diagnosis by the regulatory agencies due to the lack of verifications on large patient cohorts and suitable analytical technologies. In this paper, the effect of human blood sample handling was studied prior to N-glycosylation profiling by capillary electrophoresis, coupled with high sensitivity fluorescence detection. Special attention was paid to the preservation of sialylated structures because of their important clinical - biological relevance. Our results suggested that it is adequate to refrigerate and store the collected total blood samples prior to analysis to obtain unbiased results. Furthermore, we report on the good practice of serum sample handling in order to prevent decomposition of the sialylated structures. Our findings may promote procedure standardization and easier clinical translation of diagnostic N-glycosylation profiling in molecular medicinal applications.

Machine Learning Based Analysis of Human Serum N-glycome Alterations to Follow up Lung Tumor Surgery.

The human serum N-glycome is a valuable source of biomarkers for malignant diseases, already utilized in multiple studies. In this paper, the N-glycosylation changes in human serum proteins were analyzed after surgical lung tumor resection. Seventeen lung cancer patients were involved in this study and the N-glycosylation pattern of their serum samples was analyzed before and after the surgery using capillary electrophoresis separation with laser-induced fluorescent detection. The relative peak areas of 21 N-glycans were evaluated from the acquired electropherograms using machine learning-based data analysis. Individual glycans as well as their subclasses were taken into account during the course of evaluation. For the data analysis, both discrete (e.g., smoker or not) and continuous (e.g., age of the patient) clinical parameters were compared against the alterations in these 21 N-linked carbohydrate structures. The classification tree analysis resulted in a panel of N-glycans, which could be used to follow up on the effects of lung tumor surgical resection.

On-line enrichment of N-glycans by immobilized metal-affinity monolith for capillary electrophoresis analysis.

A novel N-glycan enrichment strategy is presented using unexpected but strong interactions between the sulfonate groups brought by the fluorescent dye of glycans and the Zr4+ modified poly(ethylene glycol methacrylate phosphate (EGMP)-co-acrylamide (AM)-co-bis-acrylamide (BAA)) monolith. The poly (EGMP-co-AM-co-BAA) monolith was synthesized via ultraviolet (UV) irradiation and then functionalized with Zr4+. The obtained monolith was characterized with scanning electron microscopy and mercury intrusion porosimetry. Large through-pores and a continuous skeleton with high permeability were observed. The N-glycans were labeled with the 1-aminopyrene-3, 6, 8-trisulfonic acid (APTS) and enriched by the Zr4+ modified monolith through IMAC interaction. This enrichment step was then coupled off-line to capillary electrophoresis (CE) separation with laser induced fluorescence (LIF) detection. Successful preconcentration of the APTS labeled maltooligosaccharide ladder was achieved under optimized conditions. Enrichment factors obtained for the maltooligosaccharides ranged from 9 to 24 with RSDs from 2.0% to 9.2% (n = 3). Moreover, very good repeatabilities (<6.7%) were obtained for glucose oligomers (4-15 glucose units) corresponding to sizes expected for N-glycans, demonstrating the great potential of this Zr4+ modified monolith to enrich APTS labeled glycans from N-glycoproteins. The proposed method was then successfully applied for the enrichment of N-glycans released from Ribonuclease B, in which case all five expected oligomannose glycans (Man 5 to Man 9) were successfully enriched. Thanks to the advantage of the method to enrich selectively APTS-glycans compared to the commercial SPE columns composed of HILIC or PGC materials, the first proof of concept of on-line enrichment coupled to CE-LIF separation was demonstrated for maltooligosaccharides as well.

Multilevel Characterization of Antibody-Ligand Conjugates by CESI-MS.

AIM: To demonstrate the capabilities of our new capillary electrophoresis - mass spectrometry method, which facilitates highly accurate relative quantitation of modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates. BACKGROUND: Antibody-drug conjugates play important roles in medical discovery for imaging and therapeutic intervention. The localization and stoichiometry of the conjugation can affect the orientation, selectivity, specificity, and strength of molecular interactions, influencing biochemical function. OBJECTIVE: To demonstrate the option to analyze the localization and stoichiometry of antibody-ligand conjugates by using essentially the same method at all levels including ligand infusion, peptide mapping, as well as reduced and intact protein analysis. MATERIALS AND METHODS: Capillary electrophoresis coupled with electrospray ionization mass spectrometry was used to analyze the antibody-ligand conjugates. RESULTS: We identified three prevalent ligand conjugation sites with estimated stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37, illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of antibody-drug conjugates. CONCLUSION: The developed multilevel analytical method offers a comprehensive way to determine the localization and stoichiometry of antibody-drug conjugates for molecular medicinal applications. In addition, a significant advantage of the reported approach is the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not common with other liquid phase separation methods such as LC.

Glycoprotein biomarkers and analysis in chronic obstructive pulmonary disease and lung cancer with special focus on serum immunoglobulin G.

Chronic obstructive pulmonary disease (COPD) and lung cancer are two major diseases of the lung with high rate of mortality, mostly among tobacco smokers. The glycosylation patterns of various plasma proteins show significant changes in COPD and subsequent hypoxia, inflammation and lung cancer, providing promising opportunities for screening aberrant glycan structures contribute to early detection of both diseases. Glycoproteins associated with COPD and lung cancer consist of highly sialylated N-glycans, which play an important role in inflammation whereby hypoxia leads to accumulation of sialyl Lewis A and X glycans. Although COPD is an inflammatory disease, it is an independent risk factor for lung cancer. Marked decrease in galactosylation of plasma immunoglobulin G (IgG) together with increased presence of sialic acids and more complex highly branched N-glycan structures are characteristic for COPD and lung cancer. Numerous glycan biomarkers have been discovered, and analysis of glycovariants associated with COPD and lung cancer has been carried out. In this paper we review fundamental glycosylation changes in COPD and lung cancer glycoproteins, focusing on IgG to provide an opportunity to distinguish between the two diseases at the glycoprotein level with diagnostic value.

N-glycosylation analysis of biopharmaceuticals by multicapillary gel electrophoresis: Generation and application of a new glucose unit database.

As glycomics research is gaining momentum in the biopharmaceutical industry, there is an increasing need for reproducible high throughput glycoanalytical methods to monitor and characterize the N-glycosylation of therapeutic glycoproteins. Since the glycosylation pattern of glycobiotherapeutics influences their important biological functions, approaches to comprehensively analyze these complex molecules is of high importance. This paper reports on the use of multicapillary gel electrophoresis in high throughput analysis of fluorophore labeled partitioned N-glycan libraries to generate a new glucose unit database that was consequently applied to identify the carbohydrate structures of two high profile biopharmaceuticals, adalimumab and etanercept.

Expanding the capillary electrophoresis-based glucose unit database of the GUcal app.

GUcal is a standalone application for automatically calculating the glucose unit (GU) values for separated N-glycan components of interest in an electropherogram and suggests their tentative structures by utilizing an internal database. We have expanded the original database of GUcal by integrating all publicly available capillary electrophoresis (CE) data in the GlycoStore collection (https://www.glycostore.org) and with in-house measured GU values. The GUcal app is freely available online (https://www.gucal.hu) and readily facilitates CE-based high throughput GU value determination for first line structural elucidation.

Comparative analysis of the human serum N-glycome in lung cancer, COPD and their comorbidity using capillary electrophoresis.

Lung cancer (LC) and chronic obstructive pulmonary disease (COPD) are prevalent ailments with a great challenge to distinguish them based on symptoms only. Since they require different treatments, it is important to find non-invasive methods capable to readily diagnose them. Moreover, COPD increases the risk of lung cancer development, leading to their comorbidity. In this pilot study the N-glycosylation profile of pooled human serum samples (90 patients each) from lung cancer, COPD and comorbidity (LC with COPD) patients were investigated in comparison to healthy individuals (control) by capillary gel electrophoresis with high sensitivity laser-induced fluorescence detection. Sample preparation was optimized for human serum samples introducing a new temperature adjusted denaturation protocol to prevent precipitation and increased endoglycosidase digestion time to assure complete removal of the N-linked carbohydrates. The reproducibility of the optimized method was <3.5%. Sixty-one N-glycan structures were identified in the pooled control human serum sample and the profile was compared to pooled lung cancer, COPD and comorbidity of COPD with lung cancer patient samples. One important finding was that no other sugar structures were detected in any of the patient groups, only quantitative differences were observed. Based on this comparative exercise, a panel of 13 N-glycan structures were identified as potential glycobiomarkers to reveal significant changes (>33% in relative peak areas) between the pathological and control samples. In addition to N-glycan profile changes, alterations in the individual N-glycan subclasses, such as total fucosylation, degree of sialylation and branching may also hold important glycobiomarker values.

Recent advances in glycoinformatic platforms for glycomics and glycoproteomics.

Protein glycosylation is the most complex and prevalent post-translation modification in terms of the number of proteins modified and the diversity generated. To understand the functional roles of glycoproteins it is important to gain an insight into the repertoire of oligosaccharides present. The comparison and relative quantitation of glycoforms combined with site-specific identification and occupancy are necessary steps in this direction. Computational platforms have continued to mature assisting researchers with the interpretation of such glycomics and glycoproteomics data sets, but frequently support dedicated workflows and users rely on the manual interpretation of data to gain insights into the glycoproteome. The growth of site-specific knowledge has also led to the implementation of machine-learning algorithms to predict glycosylation which is now being integrated into glycoproteomics pipelines. This short review describes commercial and open-access databases and software with an emphasis on those that are actively maintained and designed to support current analytical workflows.

Commentary regarding "Decision support algorithm for the selection of analytical methods in organic compounds detection for future extraterrestrial exploratory missions".

Mátyás et al. recently published their paper entitled as "Decision support algorithm for the selection of analytical methods in organic compounds detection for future extraterrestrial exploratory missions," which we found interesting. However, there are some points of the developed method that need to be refined to get a practical tool for practitioners. In this commentary, we have summarized our suggestions and comments mainly from the chemical/separation point of view.

Effect of the flow profile on separation efficiency in pressure-assisted reversed-polarity capillary zone electrophoresis of anions: Simulation and experimental evaluation.

Capillary electrophoresis connected to electrospray ionization mass spectrometry is a promising combination to analyze complex biological samples. The use of sheathless electrospray ionization interfaces, such as a porous nanoelectrospray capillary emitter, requires the application of forward flow (either by pressure or electroosmosis) to maintain the electrospray process. The analysis of solute molecules with strong negative charges (e.g., aminopyrenetrisulfonate labeled glycans) necessitates a reversed-polarity capillary electrophoresis separation mode, in which case the electroosmotic flow is counter current, thus pressure assistance is necessary. In this study, we compared the effect of forced convection with and without counter electroosmotic flow on the resulting separation efficiency in capillary electrophoresis based on flow profile simulations by computational fluid dynamics technique and by actual experiments. The efficiencies of the detected peaks were calculated from the resulting electropherograms and found approximately 950 000 plates/m for electrophoresis with counter electroosmotic flow, 20 000 plates/m with pressure only (such as would be in open tubular liquid chromatography), and 480 000 plates/m for electrophoresis with simultaneous counter electroosmotic flow and forward pressure assistance, which validates the simulation data.