On-line Coupling Research Articles

Online coupling of size exclusion chromatography to capillary-enhanced Raman spectroscopy for the identification of protein classes in hemolyzed blood serum

Online coupling of size exclusion chromatography to capillary-enhanced Raman spectroscopy for the identification of protein classes in hemolyzed blood serum

In-depth characterization of a cysteine-linked ADC disitamab vedotin by multiple LC-MS analysis methods and cutting-edge imaged capillary isoelectric focusing coupled with native mass spectrometry

The characterization of cysteine-linked antibody‒drug conjugates (ADCs) can be more challenging than that of monoclonal antibodies (mAbs) and lysine-linked ADCs because the interchain disulfide bonds are reduced for payload conjugation, and the chains are noncovalently bonded to each other. Furthermore, payload conjugation and disulfide bond reduction/scrambling may introduce additional charge heterogeneity to biomolecules. This study illustrates an innovative workflow employing multiple separation techniques and tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of disitamab vedotin, a recent-generation cysteine-linked ADC, including reversed-phase liquid chromatography (RPLC), ion exchange chromatography (IEX) and image capillary isoelectric focusing (icIEF). RPLC was employed for reduced chains analysis, subunit analysis and peptide mapping. IEX and icIEF were used for charge heterogeneity analysis. The innovation of the integrated methodology emphasizes the importance of cutting-edge icIEF-MS online coupling under near-native conditions to reveal the heterogeneity of disitamab vedotin.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

Native Proteomics by Capillary Zone Electrophoresis‐Mass Spectrometry

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid‐phase separations. Native proteomics should provide the most accurate bird’s‐eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well‐purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra‐high mass range (UHMR) Orbitrap mass spectrometer. The nCZE‐MS technique enabled the measurement of a 115‐kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE‐MS analysis of an E.coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30‐400 kDa in a single run while consuming only 50‐ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

A comprehensive study on solvent effect and establishment of n-hexane solvent system based normal-phase liquid chromatography × reversed-phase liquid chromatography for isolation of natural products

Reversed-phase liquid chromatography (RPLC) represents an effective separation method, and is widely employed as the second dimension in most 2D-LC systems. Nevertheless, the solvent effect of the eluent from the first dimension on RPLC presents a challenge to the online coupling of RPLC with other separation modes, particularly normal phase liquid chromatography (NPLC). To address this issue, a comprehensive understanding of the solvent effect is essential. Following a comprehensive investigation into the influence of diverse solvents on RPLC separations, it was observed that alkane solvents, such as n-hexane, exhibited a pronounced tendency to be retained during RPLC separations. Such solvents do not affect the analysis of samples with weaker retention abilities than themselves, even when a large injection volume is used. The solvent effect was thus reduced by employing n-hexane-based solvent dilution. Leveraging the markedly enhanced solvent tolerance and extensive injection volume in RPLC, a versatile integration of the NPLC and RPLC was devised, necessitating merely a purge pump and a 10 port 2 position valve in conjunction with two sample loops. The novel 2D-LC system was then deployed for the analysis of propolis, a naturally occurring complex sample, and demonstrated remarkable separation efficiency.

Native Proteomics by Capillary Zone Electrophoresis-Mass Spectrometry.

Native proteomics measures endogenous proteoforms and protein complexes under a near physiological condition using native mass spectrometry (nMS) coupled with liquid-phase separations. Native proteomics should provide the most accurate bird's-eye view of proteome dynamics within cells, which is fundamental for understanding almost all biological processes. nMS has been widely employed to characterize well-purified protein complexes. However, there are only very few trials of utilizing nMS to measure proteoforms and protein complexes in a complex sample (i.e., a whole cell lysate). Here, we pioneer the native proteomics measurement of large proteoforms or protein complexes up to 400 kDa from a complex proteome via online coupling of native capillary zone electrophoresis (nCZE) to an ultra-high mass range (UHMR) Orbitrap mass spectrometer. The nCZE-MS technique enabled the measurement of a 115-kDa standard protein complex while consuming only about 0.1 ng of protein material. nCZE-MS analysis of an E . coli cell lysate detected 72 proteoforms or protein complexes in a mass range of 30-400 kDa in a single run while consuming only 50-ng protein material. The mass distribution of detected proteoforms or protein complexes agreed well with that from mass photometry measurement. This work represents a technical breakthrough in native proteomics for measuring complex proteomes.

A combination of multiple LC-MS approaches for the comprehensive characterization of cysteine-linked ADCs

Antibody-drug conjugates (ADCs) represent the forefront of the next generation of biopharmaceuticals. An ADC typically comprises an antibody covalently linked to a cytotoxic drug via a linker, resulting in a highly heterogeneous product. This study focuses on the analysis of a custom-made cysteine-linked ADC. Initially, we developed a LC-MS-based characterization workflow using brentuximab vedotin (Adcetris®), encompassing native intact MS, analysis of reduced chains and subunits under denaturing condition, peptide mapping and online strong cation exchange chromatography coupled with UV and mass spectrometry detection (SCX-UV-MS) applied for brentuximab vedotin first time reported. Subsequently, we applied this in-depth characterization workflow to a custom-made cysteine-linked ADC. The measured drug-to-antibody ratio(DAR) of this ADC is 6.9, further analysis shown that there is a small amount of unexpected over-conjugation. Over-conjugation sites were successfully identified using multiple UHPLC-MS based characterization techniques. Also, one competitively cysteine-conjugated impurity was observed in native intact MS results, by combing native intact MS, reduced chains, subunit analysis and peptide mapping results, the impurity conjugation sites were also identified. Since this molecule is at early development stage, this provides important information for conjugation process improvement and link-drug material purification. SCX-UV-MS approach can separate the custom-made cysteine-linked ADC carrying different payloads and reduce the complexity of the spectra. The integrated approach underscores the significance of combining the SCX-UV-MS online coupling technique with other characterization methods to elucidate the heterogeneity of cysteine-linked ADCs.

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

Integrated Optimization of Train Diagrams and Rolling Stock Circulation with Full-Length and Short-Turn Routes of Virtual Coupling Trains in Urban Rail Transit

The advancement of virtual coupling technology in urban rail transit has facilitated the online coupling and decoupling of trains, enabling a range of flexible transportation configurations, including various route types and adjustable formations. This study targets the fluctuating passenger demands on urban rail lines, aiming to minimize both passenger travel and operational costs. The model integrates constraints associated with virtual coupling, train operations, rolling stock circulation, and the interaction between virtually coupled trains and passenger arrivals. New decision variables are introduced to depict the train formation state under virtual coupling scenarios. An integrated optimization model for train diagrams and rolling stock circulation under virtual coupling conditions is developed, employing a genetic-simulated annealing algorithm informed by train operation simulations. A case study on an urban rail line during the morning peak examines the optimization of train diagrams for full-length and short-turn routes. Findings confirm that virtual coupling technology effectively adapts to lines with uneven passenger flow distribution, significantly enhancing the match between supply and demand, equalizing spatial and temporal traffic variations, and harmonizing the quality of passenger services with operational efficiency.

Application of on-line sample preconcentration by large-volume dual preconcentration by isotachophoresis and stacking (LDIS) on straight-channel microchips.

In this study, large-volume dual preconcentration by isotachophoresis and stacking (LDIS) which is an on-line sample preconcentration technique coupling large-volume sample stacking with an electroosmotic flow pump (LVSEP) with transient isotachophoresis (tITP) was applied to microchip electrophoresis (MCE) for improving both detection sensitivities and peak shapes. To realize LDIS in MCE, we investigated experimental procedures for injecting a short plug of a leading electrolyte (LE) solution into a straight microchannel without any sophisticated injector apparatus. We found that a short LE plug could be injected into a sample-filled straight-channel only by making the liquid level of the LE solution in an outlet reservoir higher than that in an inlet one. By applying a reversed-polarity voltage to the microchip, anionic analytes injected throughout the microchannel were first enriched by LVSEP, followed by tITP. Through the second preconcentration effect by tITP in LDIS, sensitivity enhancement factor (SEF) and asymmetry factor for a standard dye were improved from 878 and 0.62 to 1330 and 1.14, respectively, relative to those in conventional LVSEP. It should be noted that more viscous running buffer containing sieving polymers could be employed to the LDIS analysis, which was effective for improving the SEF and the separation efficiencies, especially for bio-polymeric compounds. Finally, LDIS was applied to the oligosaccharide and protein analyses in MCE, resulting in the SEFs of 1410 and ca. 50 for maltotriose and bovine milk casein, respectively.

Development of Noninvasive Method for the Automated Analysis of Nine Steroid Hormones in Human Saliva by Online Coupling of In-Tube Solid-Phase Microextraction with Liquid Chromatography–Tandem Mass Spectrometry

Accurate measurement of steroid hormones is crucial to elucidate new mechanisms of action and diagnose steroid metabolism-related diseases. This study presents a simple, sensitive, and automated analytical method for nine representative steroid hormones. The method involves on-line coupling of in-tube solid-phase microextraction (IT-SPME) with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The steroid hormones were extracted and enriched on a Supel-Q PLOT capillary column using IT-SPME. Subsequently, they were separated and detected within 6 min using a Discovery HS F5-3 column and positive ion mode multiple reaction monitoring system via LC–MS/MS. Calibration curves of these compounds using each stable isotope-labeled internal standard (IS) showed linearity with correlation coefficients greater than 0.9990 in the range of 0.01–40 ng/mL, with limits of detection (S/N = 3) of 0.7–21 pg/mL. Moreover, intra- and inter-day variations were lower than 8.1 and 15% (n = 6), respectively. The recoveries of these compounds from saliva samples were in the range of 82–114%. The developed IT-SPME/LC–MS/MS method of steroid hormones is a highly sensitive, specific, and non-invasive analytical method that allows extraction and enrichment with no organic solvents, and enables direct automated online analysis by simply ultrafiltrating a small sample of saliva.

What is the relative impact of nudging and online coupling on meteorological variables, pollutant concentrations and aerosol optical properties?

Abstract. Meteorological and chemical modelling at the regional scale often involve the nudging of the modelled meteorology towards reanalysis fields and meteo-chemical coupling to properly consider the interactions between aerosols, clouds and radiation. Both types of processes can change the meteorology, but not for the same reasons and not necessarily in the same way. To assess the possible interactions between nudging and online coupling, several simulations are carried out with the WRF–CHIMERE (Weather Research and Forecasting) model in its offline and online configurations. Through comparison with measurements, we show that the use of nudging significantly improves the model performances. We also show that coupling changes the results much less than nudging. Finally, we show that when nudging is used, it limits the variability in the results due to coupling.

Research of saccharides and related biocomplexes: A review with recent techniques and applications.

Saccharides and biocompounds as saccharide (sugar) complexes have various roles and biological functions in living organisms due to modifications via nucleophilic substitution, polymerization, and complex formation reactions. Mostly, mono-, di-, oligo-, and polysaccharides are stabilized to inactive glycosides, which are formed in metabolic pathways. Natural saccharides are important in food and environmental monitoring. Glycosides with various functionalities are significant in clinical and medical research. Saccharides are often studied with the chromatographic methods of hydrophilic interaction liquid chromatography and anion exchange chromatograpy, but also with capillary electrophoresis and mass spectrometry with their on-line coupling systems. Sample preparation is important in the identification of saccharide compounds. The cases discussed here focus on bioscience, clinical, and food applications.

Online coupling of liquid chromatography and two-dimensional diffusion ordered spectroscopy for the analysis of oligostyrenes

The aim of this study was to introduce a powerful coupling of Liquid Adsorption Chromatography (LAC) and Diffusion-Ordered Spectroscopy (DOSY) for comprehensive structure analysis. This new hyphenation approach facilitated the simultaneous separation of a polymer mixture and the determination of molar masses within a single 3D experiment. The online coupling of High-Performance Liquid Chromatography (HPLC) and two-dimensional DOSY-NMR will be called 3D-LAC-NMR-DOSY experiment. Our methodology enabled the chromatographic separation of analytes based on their chemical heterogeneity, and provided accurate molar masses of the analytes through 2D-DOSY. This new method was demonstrated on a polystyrene oligomer mixture. In this case, the oligostyrenes could be separated with LAC according to their tacticity and chain length in protonated acetonitrile as eluent and DOSY measurements provided the molar masses of each oligomer. In order to show the power of the 3D-LAC-NMR-DOSY method, the comparison to 2D-DOSY, 3D-DOSY and LAC-NMR was separately evaluated. Furthermore, the recently published solvent-independent molar mass calibration of diffusion coefficients was also successfully applied in our LAC-DOSY studies for molar mass predictions of the oligomers in acetonitrile. The predicted molar masses were in good agreement with the LAC-DOSY measurements and were verified by calibrations of diffusion coefficients and mass spectrometry. Finally, this pioneering 3D technique offers a powerful new tool for advancing structure analysis and enhancing our understanding of complex systems such as oligostyrenes.

Mass spectrometry study on SARS-CoV-2 recombinant vaccine with comprehensive separation techniques to characterize complex heterogeneity

SARS-CoV-2, the causative agent of COVID-19, has imposed a major public health threat, which needs effective therapeutics and vaccination strategies. Several potential candidate vaccines being rapidly developed are in clinical evaluation and recombinant vaccine has gained much attention thanks to its potential for greater response predictability, improved efficacy, rapid development and reduced side effects. Recombinant vaccines are designed and manufactured using bacterial, yeast cells or mammalian cells. A small piece of DNA is taken from the virus or bacterium against which we want to protect and inserted into the manufacturing cells. Due to the extremely complex heterogeneity of SARS-CoV-2 recombinant vaccine, single technology platform cannot achieve thorough and accurate characterization of such difficult proteins so integrating comprehensive technologies is essential. This study illustrates an innovative workflow employing multiple separation techniques tandem high-resolution mass spectrometry for comprehensive and in-depth characterization of SARS-CoV-2 recombinant vaccine, including ultra-high performance liquid chromatography (UHPLC), ion exchange chromatography (IEX) and imaged capillary isoelectric focusing (icIEF). The integrated methodology focuses on the importance of cutting-edge icIEF-MS online coupling and icIEF fractionation applied to revealing the heterogeneity secret of SARS-CoV-2 recombinant vaccine.

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.

Online coupling of miniaturized HPLC and high performance thin layer chromatography by a fractionation unit for effect directed analysis

The rising discharge of anthropogenic chemicals into aquatic environments poses a significant threat, necessitating effective monitoring strategies. This study introduces an innovative approach to effect-directed analysis (EDA) by coupling liquid chromatography (LC) with high-performance thin-layer chromatography (HPTLC), utilizing a modified MALDI spotter. The objective is to optimize fractionation parameters for sample application and assess the method's viability in identifying acetylcholinesterase (AChE) inhibitors, specifically malathion, parathion, and chlorfenvinphos.The optimization process involves controlling sample volume, spot shape, and spot distances on HPTLC plates. Successful application is demonstrated by a miniaturized LC system coupled to the HPTLC plate via spotter, allowing effective separation and identification of AChE inhibitors. The study further explores the method's application to water samples from a river with predominantly agricultural drainage area.Spiked samples reveal distinct active spots, identified through extraction and subsequent high-resolution mass spectrometry (HRMS) measurements. However, results indicate the absence of AChE inhibitors in non-spiked water samples, affirming the efficacy of the EU ban on most organophosphate pesticides. The usefulness of HPTLC in separation of coeluting substances from HPLC is emphasized, demonstrating its suitability for the effect-directed analysis of complex samples.This work shows the integration of HPTLC with liquid chromatography for EDA, offering a powerful tool for identifying and monitoring AChE inhibitors in water samples. The approach addresses limitations in current monitoring strategies and provides insights into the presence and impact of chemicals in aquatic ecosystems. The study contributes to ongoing efforts to enhance water quality monitoring, aligning with the principles of the EU Water Framework Directive.

Coupling of droplet-on-demand microfluidcs with ESI/MS to study single-cell catalysis.

Droplet microfluidics provides an efficient method for analysing reactions within the range of nanoliters to picoliters. However, the sensitive, label-free and versatile detection with ESI/MS poses some difficulties. One challenge is the difficult association of droplets with the MS signal in high-throughput droplet analysis. Hence, a droplet-on-demand system for the generation of a few droplets can address this and other problems such as surfactant concentration or cross-contamination. Accordingly, the system has been further developed for online coupling with ESI/MS. To achieve this, we developed a setup enabling on-demand droplet generation by hydrodynamic gating, with downstream microscopic droplet detection and MS analysis. This facilitated the incorporation of 1-9 yeast cells into individual 1-5 nL droplets and the monitoring of yeast-catalysed transformation from ketoester to ethyl-3-hydroxybutyrate by MS. With our method a mean production rate of 0.035 ± 0.017 fmol per cell per h was observed with a detection limit of 0.30 μM. In conclusion, our droplet-on-demand method is a versatile and advantageous tool for cell encapsulation in droplets, droplet imaging and reaction detection using ESI/MS.

Online Coupling of Size Exclusion Chromatography to Capillary Enhanced Raman Spectroscopy for the Analysis of Proteins and Biopharmaceutical Drug Products.

The online coupling of size exclusion chromatography (SEC) to capillary enhanced Raman spectroscopy (CERS) based on a liquid core waveguide (LCW) flow cell was applied for the first time to assess the higher-order structure of different proteins. This setup allows recording of Raman spectra of the monomeric protein within complex mixtures, since SEC enables the separation of the monomeric protein from matrix components such as excipients of a biopharmaceutical product and higher molecular weight species (e.g., aggregates). The acquired Raman spectra were used for structural elucidation of well characterized proteins such as bovine serum albumin, hen egg white lysozyme, and β-lactoglobulin and of the monoclonal antibody rituximab in a medicinal product. Additionally, the CERS detection of the disaccharide sucrose, which is used as a stabilizing excipient, was quantified to achieve a limit of detection (LOD) of 120 μg and a limit of quantification (LOQ) of 363 μg injected on the column.

Sol-gel immunoaffinity chromatography for isolation of deoxynivalenol and zearalenone from food and environmental samples

ABSTRACT The presence of mycotoxins in food is a significant concern due to their potential health risks. It is estimated that around 25% of global food production is contaminated with mycotoxins, which can be produced by certain types of fungi that infect crops during different stages of growth. As a result, it is crucial to monitor mycotoxins in food sources and develop effective methods for removal or reduction. One technique commonly used for detecting and separating mycotoxins is immunoaffinity chromatography (IAC). Traditionally, IAC uses antibodies covalently bonded to a solid support material, but this can lead to conformational changes and reduced affinity towards the analytes. To address these limitations, researchers have explored alternative methods for immobilising biomolecules, such as the sol-gel method, which involves encapsulating biomolecules within the pores of a glass matrix. This approach makes it easier to reuse the immunoaffinity columns and reduces the need for bacteriostatic agents, making the process more cost-effective and efficient. The results showed that sol-gel IAS was able to effectively separate these toxins from complex matrices, demonstrating its potential for widespread application in food safety monitoring. Additionally, the ability of sol-gel IAS to remove mycotoxins from environmental samples tested was highlighting its versatility and potential for broader applications beyond food safety. Finally, they demonstrated the compatibility of sol-gel IAS with high-performance liquid chromatography, enabling online coupling of the two techniques for improved efficiency and accuracy. The findings of this study suggest that sol-gel IAC is a promising method for the detection and separation of mycotoxins in various matrices, including food, environmental samples, and other biological fluids. Its advantages over traditional IAC methods make it an attractive option for large-scale analysis and monitoring of mycotoxins in diverse settings.