In Migratio Noncovalent Fluorophore Labeling of Proteins by Propidium Iodide in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

Sodium dodecyl sulfate capillary gel electrophoresis is one of the frequently used methods for size-based protein separation in molecular biology laboratories and the biopharmaceutical industry. To increase throughput, quite a few multicapillary electrophoresis systems have been recently developed, but most of them only support fluorescence detection, requiring fluorophore labeling of the sample proteins. To avoid the time-consuming derivatization reaction, we developed an on-column labeling approach utilizing propidium iodide for the first time in SDS-CGE of proteins, a dye only used before for nucleic acid analysis. As a key ingredient of the gel-buffer system, the oppositely migrating positively charged propidium ligand in migratio complexes with the SDS-proteins, therefore, supports in situ labeling during the electrophoretic separation process, not requiring any extra pre- or postcolumn derivatization step. A theoretical treatment is given to shed light on the basic principles of this novel online labeling process, also addressing the influence of propidium iodide on the electroosmotic flow, resulting in reduced retardation. The concept of propidium labeling in SDS-CGE was first demonstrated using a commercially available protein sizing ladder ranging from 6.5 to 200 kDa with different isoelectric points and post-translational modifications. Considering the increasing number of protein therapeutics on the market next, we focused on the labeling optimization of a therapeutic monoclonal antibody and its subunits, including the addition of the nonglycosylated heavy chain. Peak efficiency and resolution were compared between noncovalent and covalent labeling. The effect of ligand concentration on the effective and apparent electrophoretic mobility, the resulting peak area, and the resolution were all evaluated in view of the theoretical considerations. The best detection sensitivity for the intact monoclonal antibody was obtained by using 200 µg/mL propidium iodide in the separation medium (LOD 2 µg/mL, 1.35 × 10-8 M) with excellent detection linearity over 3 orders of magnitude. On the other hand, the resolution between the biopharmaceutical protein test mixture components containing the intact and subunit fragments of the therapeutic monoclonal antibody was very good in the ligand concentration range of 50-200 µg/mL, but using the local maximum at 100 µg/mL for the nonglycosylated/glycosylated heavy chain pair is recommended. The figures of merit, including precision, sensitivity, detection linear range, and resolution for a sample mixture in hand, can be optimized by varying the propidium iodide concentration in the gel-buffer system, as demonstrated in this paper.

Analysis of Peptides and Proteins by Native and SDS Capillary Gel Electrophoresis Coupled to Electrospray Ionization Mass Spectrometry via a Closed-Circuit Coaxial Sheath Flow Reactor Interface.

A simple and widely applicable coaxial sheath flow reactor interface (CSFRI) is introduced for easy and robust connection of liquid-phase microseparation methods to mass spectrometric detection, especially for capillary gel electrophoresis analysis of proteins and peptides including SDS-protein complexes. The interface readily accommodated post-column reactions prior to MS detection. It was demonstrated that this novel closed-circuit connection allowed the utilization of non-MS friendly buffer components without significant ion suppression and supported stable electrospray. In SDS capillary agarose gel electrophoresis mode, addition of γ-cyclodextrin to the sheath liquid efficiently removed the SDS content of the sample and the background electrolyte in the flow reactor section by inclusion complexation, while maintaining good separation efficiency and decreasing ion suppression.

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.

Size separation of sodium dodecyl sulfate-proteins by capillary electrophoresis in dilute and ultra-dilute dextran solutions.

SDS capillary gel electrophoresis is a widely used in the biopharma and the biomedical fields for rapid size separation of proteins. However, very limited information is available on the use of dilute and ultra-dilute sieving matrices for SDS-protein analysis. Here, background electrolytes (BGEs) containing 1%-0% dextran were used in borate-based BGE to separate a protein sizing ladder (PSL) ≤225 kDa and the intact and subunit forms of a therapeutic monoclonal antibody (mAb). The separation performance for the PSL and mAb components differed significantly with decreasing dextran concentration. Ferguson and reptation plots were used to elucidate the separation mechanism. Highly diluted dextran solutions resulted in linear Ferguson plots for both solute types (cf. Ogston theory) in spite of this model assumes a rigid pore structure, thus cannot describe the separation mechanism in ultra-dilute polymer solutions with no reticulations. The saddle differences between the resolution of the PSL and the intact/subunit mAb forms in ultra-dilute dextran-borate matrices suggested the importance of shape selectivity, manifested by the adequate separation of the SDS covered intact as well as light and heavy chain subunits of the therapeutic mAb even at zero dextran concentration.

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.

Electromigration Dispersion in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Proteins.

The electromigration dispersion of the light- and heavy-chain subunit peaks of the therapeutic monoclonal antibody omalizumab was investigated in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran sieving matrices. Increasing boric acid content (340-640 mM) caused electromigration dispersion shifts for both low (2%)- and high (10%)-dextran-concentration gels in all gel-buffer compositions. In case of the heavy-chain fragment, elevated borate concentrations resulted in decreasing tailing and increasing fronting with the use of higher- and lower-dextran-concentration gels, respectively. The light-chain fragment, on the other hand, exhibited increased fronting with increasing borate concentration for both dextran concentrations examined in this study. Increase of the glycerol ingredient level in the gel-buffer system caused the same effect as the increasing borate concentration in both dextran concentrations. The detected electromigration dispersion was considered as the result of the formation of monomeric and dimeric glycerol-borate complexes as co-ionic constituents, migrating slower than that of the unconjugated tetrahydroxyborate. In addition, complexation of the tetrahydroxyborate anion with the glucose building blocks of the dextran polymer decreased its mobility to practically zero, contributing to further decrease in the resultant effective mobility of the co-ionic species. We suggest that the observed fronting and/or tailing peak shapes of the monoclonal antibody fragments in SDS-CGE at increasing boric acid concentrations can be considered as the result of multiple effects including changes in pH, sieving matrix pore size, viscosity, and the mobility variation of the co-ionic borate adducts with the gel-buffer ingredients. While electromigration dispersion-mediated band broadening, in general, can be minimized via matching the effective mobility of the co-ionic species to the analyte molecules of interest, in case of borate cross-linked dextran gels, optimization of the boric acid concentration required special consideration of its gel cross-linking function. For the light- and heavy-chain fragments of the IgG analyte, best peak shapes were attained with the use of 10% dextran/340 mM boric acid and 10% dextran/640 mM boric acid-containing gel-buffer systems, respectively. Based on this observation, here we introduce the concept of borate-gradient-mediated transient mobility matching in SDS-CGE of proteins. This novel approach resulted in close to optimal peak shapes for the distantly migrating IgG subunits within a single run, as well as unraveled the long-sought possible solution to perform capillary pore-size-gradient gel electrophoresis.

Applications of capillary electrophoresis for biopharmaceutical product characterization.

Capillary electrophoresis (CE), after being introduced several decades ago, has carved out a niche for itself in the field of analytical characterization of biopharmaceutical products. It does not only offer fast separation, high resolution in miniaturized format, but equally importantly represents an orthogonal separation mechanism to high-performance liquid chromatography. Therefore, it is not surprising that CE-based methods can be found in all major pharmacopoeias and are recommended for the analysis of biopharmaceutical products during process development, characterization, quality control, and release testing. Different separation formats of CE, such as capillary gel electrophoresis, capillary isoelectric focusing, and capillary zone electrophoresis are widely used for size and charge heterogeneity characterization as well as purity and stability testing of therapeutic proteins. Hyphenation of CE with MS is emerging as a promising bioanalytical tool to assess the primary structure of therapeutic proteins along with any impurities. In this review, we confer the latest developments in capillary electrophoresis, used for the characterization of critical quality attributes of biopharmaceutical products covering the past 6 years (2015-2021). Monoclonal antibodies, due to their significant share in the market, have been given prioritized coverage.

Evaluation of an icIEF-MS system for comparable charge variant analysis of biotherapeutics with rapid peak identification by mass spectrometry.

Protein therapeutics are usually produced in heterogeneous forms during bioproduction and bioprocessing. Heterogeneity results from post-translational modifications that can yield charge variants and require characterization throughout product development and manufacturing. Isoelectric focusing (IEF) with UV detection is one of the most common methods to evaluate protein charge heterogeneity in the biopharmaceutical industry. To identify charge variant peaks, a new imaged microfluidic chip-based isoelectric focusing (icIEF) system coupled directly to mass spectrometry was recently reported. Bridging is required to demonstrate comparability between existing and new technology. As such, here we demonstrate the comparability of the pI value measurement and relative charge species distributions between the icIEF-MS system and the control data from a frequently utilized methodology in the biopharmaceutical industry for several blinded development-phase biopharmaceutical monoclonal antibodies across a wide pI range of 7.3-9.0. Hyphenation of the icIEF system with mass spectrometry enabled direct and detailed structural determination of a test molecule, with masses suggesting acidic and basic shifts are caused by sialic acid additions and the presence of unprocessed lysine residues. In addition, MS analysis further identified several low-abundance glycoforms. The icIEF-MS system provides sample quantification, characterization, and identification of mAb proteoforms without sacrificing icIEF quantification comparability or speed.

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.

Effect of the Monomer Cross-Linker Ratio on the Separation Selectivity of Monoclonal Antibody Subunits in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis.

With the increasing interest in the biopharmaceutical industry toward novel and innovative protein therapeutics, improved separation techniques are important, especially for the analysis of highly glycosylated candidates. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using borate cross-linked dextran is one of the most frequently used methods to analyze biotherapeutic proteins in process control as well as in release and stability testing. In this work, the effect of the monomer (dextran) and cross-linker (borate) ratio was studied in SDS-CGE analysis of a therapeutic monoclonal antibody test item in its reduced and intact forms. A retention model was developed for better understanding of the separation selectivity between the non-glycosylated and glycosylated heavy chain fragments, exploiting the interaction between the dextran-borate adducts and the glycan moiety of the therapeutic antibody. The monomer cross-linker ratio played a significant role in the overall analysis times and affected the separation selectivity between the non-glycosylated and regular (glycosylated) heavy chain fragments; however, it had no effect on the separation of the regular and non-glycosylated intact forms of the monoclonal antibody. Introduction of three-dimensional selectivity plots offered an easy separation optimization option for the separation problem in hand.

Fundamentals of Capillary Electrophoretic Migration and Separation of SDS Proteins in Borate Cross-Linked Dextran Gels.

Recent progress in the development and production of new, innovative protein therapeutics require rapid and adjustable high-resolution bioseparation techniques. Sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) using a borate (B) cross-linked dextran (D) separation matrix is widely employed today for rapid consistency analysis of therapeutic proteins in manufacturing and release testing. Transient borate cross-linking of the semirigid dextran polymer chains leads to a high-resolution separation gel for SDS-protein complexes. To understand the migration and separation basis of the D/B gel, the present work explores various gel formulations of dextran monomer (2, 5, 7.5, and 10%) and borate cross-linker (2 and 4%) concentrations. Ferguson plots were analyzed for a mixture of protein standards with molecular weights ranging from 20 to 225 kDa, and the resulting nonlinear concave curves pointed to nonclassical sieving behavior. While the 2% D/4% B gel resulted in the fastest analysis time, the 10% D/2% B gel was found to produce the greatest separation window, even higher than with the 10% D/4% B gel, due to a significant increase in the electroosmotic flow of the former composition in the direction opposite to SDS-protein complex migration. The study then focused on SDS-CGE separation of a therapeutic monoclonal antibody and its subunits. A combination of molecular weight and shape selectivity as well as, to a lesser extent, surface charge density differences (due to glycosylation on the heavy chain) influenced migration. Greater molecular weight selectivity occurred for the higher monomer concentration gels, while improved glycoselectivity was obtained using a more dilute gel, even as low as 2% D/2% B. This latter gel took advantage of the dextran-borate-glycoprotein complexation. The study revealed that by modulating the dextran (monomer) and borate (cross-linker) concentration ratios of the sieving matrix, one can optimize the separation for specific biopharmaceutical modalities with excellent column-to-column, run-to-run, and gel-to-gel migration time reproducibilities (<0.96% relative standard deviation (RSD)). The widely used 10% dextran/4% borate gel represents a good screening option, which can then be followed by a modified composition, optimized for a specific separation as necessary.

Determination of complex type free, non-conjugated oligosaccharide glucose unit values in tomato xylem sap for early detection of nutrient deficiency.

Although knowledge on glycan biosynthesis and processing is continuously maturing, there are still a limited number of studies that examine biological functions of N-glycan structures in plants, which remain virtually unknown. Here, the statistical correlation between nutrient (nitrogen) deficiency symptoms of crops and changes in 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled complex type free oligosaccharides is reported. While deficiency symptoms are predicted by multispectral images and Kjeldahl digestion, APTS-labeled complex type free oligosaccharides are identified by their glucose unit (GU) values in tomato xylem sap, using capillary electrophoresis with laser induced fluorescence detection (CE-LIF). Given the limited number of structures obtained from plants, archived in the literature, in the future, it is intended to create an open access database of promising indicators, namely, glycan structures that are presumably responsible for the nutrient deficiency caused stress in plants (http://glycoplants.org).

Rapid capillary gel electrophoresis analysis of human milk oligosaccharides for food additive manufacturing in-process control.

Industrial production of human milk oligosaccharides (HMOs) represents a recently growing interest since they serve as key ingredients in baby formulas and are also utilized as dietary supplements for all age groups. Despite their short oligosaccharide chain lengths, HMO analysis is challenging due to extensive positional and linkage variations. Capillary gel electrophoresis primarily separates analyte molecules based on their hydrodynamic volume to charge ratios, thus, offers excellent resolution for most of such otherwise difficult-to-separate isomers. In this work, two commercially available gel compositions were evaluated on the analysis of a mixture of ten synthetic HMOs. The relevant respective separation matrices were then applied to selected analytical in-process control examples. The conventionally used carbohydrate separation matrix was applied for the in-process analysis of bacteria-mediated production of 3-fucosyllactose, lacto-N-tetraose, and lacto-N-neotetraose. The other example showed the suitability of the method for the in vivo in-process control of a shake flask and fermentation approach of 2'-fucosyllactose production. In this latter instance, borate complexation was utilized to efficiently separate the 2'- and 3-fucosylated lactose positional isomers. In all instances, the analysis of the HMOs of interest required only a couple of minutes with high resolution and excellent migration time and peak area reproducibility (average RSD 0.26% and 3.56%, respectively), features representing high importance in food additive manufacturing in-process control.

Sample Preparation Scale-Up for Deep N-glycomic Analysis of Human Serum by Capillary Electrophoresis and CE-ESI-MS.

We introduce an efficient sample preparation workflow to facilitate deep N-glycomics analysis of the human serum by capillary electrophoresis with laser induced fluorescence (CE-LIF) detection and to accommodate the higher sample concentration requirement of electrospray ionization mass spectrometry connected to capillary electrophoresis (CE-ESI-MS). A novel, temperature gradient denaturing protocol was applied on amine functionalized magnetic bead partitioned glycoproteins to circumvent the otherwise prevalent precipitation issue. During this process, the free sugar content of the serum was significantly decreased as well, accommodating enhanced PNGase F mediated release of the N-linked carbohydrates. The liberated oligosaccharides were tagged with aminopyrene-trisulfonate, utilizing a modified evaporative labeling protocol. Processing the samples with this new workflow enabled deep CE-LIF analysis of the human serum N-glycome and provided the appropriate amount of material for CE-ESI-MS analysis in negative ionization mode.

Capillary Electrophoresis-Based N-Glycosylation Analysis in the Biomedical and Biopharmaceutical Fields.

Glycomics has a growing interest in the biopharmaceutical industry and biomedical research requiring new high-performance and high-sensitivity bioanalytical tools. Analysis of N-glycosylation is very important during the development of protein therapeutics and it also plays a key role in biomarker discovery. The most frequently used glycoanalytical methods are capillary electrophoresis, liquid chromatography, and mass spectrometry. In this chapter, the capillary electrophoresis-based N-linked carbohydrate analysis methods are conferred with emphasis on its use in the biopharmaceutical and biomedical fields.

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.

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.

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.

The Effect of Temperature in Sodium Dodecyl Sulfate Capillary Gel Electrophoresis of Protein Therapeutics.

The temperature-dependent migration of molecular weight protein size standards and several biotherapeutic proteins were studied in sodium dodecyl sulfate capillary gel electrophoresis (SDS-CGE) in the interval from 15 to 60 °C using borate cross-linked dextran sieving matrix. Arrhenius plots were generated to calculate the respective activation energy values for the various solute molecules. SDS-CGE analysis of the biotherapeutic protein test mixture revealed no correlation between the activation energy requirement of the different species and their molecular weights, emphasizing the importance of separation temperature optimization to obtain high resolution between the solute molecules of interest. In contrast, the molecular weight protein size ladder ranging from 10 to 225 kDa, built from the same polypeptide blocks with no post-translational and other modifications, showed predictable activation energy requirement. The electrophoretic mobility of the SDS-protein complexes was found to be the function of the reciprocal sixth root of the molecular weight (Mw-1/6), implying cylindrical conformation.

Capillary Electrophoresis-Mass Spectrometry at Trial by Metabo-Ring: Effective Electrophoretic Mobility for Reproducible and Robust Compound Annotation.

Capillary zone electrophoresis-mass spectrometry (CE-MS) is a mature analytical tool for the efficient profiling of (highly) polar and ionizable compounds. However, the use of CE-MS in comparison to other separation techniques remains underrepresented in metabolomics, as this analytical approach is still perceived as technically challenging and less reproducible, notably for migration time. The latter is key for a reliable comparison of metabolic profiles and for unknown biomarker identification that is complementary to high resolution MS/MS. In this work, we present the results of a Metabo-ring trial involving 16 CE-MS platforms among 13 different laboratories spanning two continents. The goal was to assess the reproducibility and identification capability of CE-MS by employing effective electrophoretic mobility (µeff) as the key parameter in comparison to the relative migration time (RMT) approach. For this purpose, a representative cationic metabolite mixture in water, pretreated human plasma, and urine samples spiked with the same metabolite mixture were used and distributed for analysis by all laboratories. The µeff was determined for all metabolites spiked into each sample. The background electrolyte (BGE) was prepared and employed by each participating lab following the same protocol. All other parameters (capillary, interface, injection volume, voltage ramp, temperature, capillary conditioning, and rinsing procedure, etc.) were left to the discretion of the contributing laboratories. The results revealed that the reproducibility of the µeff for 20 out of the 21 model compounds was below 3.1% vs 10.9% for RMT, regardless of the huge heterogeneity in experimental conditions and platforms across the 13 laboratories. Overall, this Metabo-ring trial demonstrated that CE-MS is a viable and reproducible approach for metabolomics.