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Background: Monoclonal antibodies (mAbs) undergo multiple post-translational modifications (PTMs) during production and storage, resulting for instance in charge and oxidized variants. PTMs need to be assessed as critical quality attributes to assure protein quality and safety. Capillary zone electrophoresis (CZE) enables efficient charge-based separation. The CZE method developed by He et al. (2011) is currently applied routinely in the pharmaceutical industry for profiling charge heterogeneity of mAbs. However, as the method relies on a non-volatile background electrolyte (BGE), it cannot be directly hyphenated with mass spectrometry (MS), hampering the identification of separated charge variants., Results: This study presents a CZE-UV/MS method using a neutral static capillary coating of hydroxypropyl methylcellulose combined with a volatile BGE at pH 5.0 to allow for MS-compatible mAb charge variant separations. The effect of several parameters, including pH and concentration of the BGE, applied voltage, and injected mAb concentrations on separation performance was investigated using a panel of commercially available mAbs. The optimized method was evaluated with IgG 1 and IgG 4 mAbs of varying pI (7.4-9.2) and degrees of heterogeneity. Basic and acidic variants were separated from the parent mAb using a BGE of 50 mM acetic acid adjusted to pH 5.0 with ammonium hydroxide. The relative abundances of charge variants determined with the new method showed a good correlation with the corresponding relative levels obtained with the method of He et al. CZE-MS coupling was accomplished using the nanoCEasy, a low-flow sheath liquid interface, which enabled the identification and quantitation of basic, acidic, and incomplete pyroglutamate variants, and glycoforms of the tested mAbs., Significance: This manuscript describes a new CZE-MS method that permits heterogeneity assessment of mAbs under MS-compatible conditions, providing charge variant separation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Early-stage cell line screening is a vital step in developing biosimilars of therapeutic monoclonal antibodies (mAbs). While the quality of the manufactured antibodies is commonly assessed by charge-based separation methods employing UV absorbance detection, these methods lack the ability to identify resolved mAb variants. We evaluated the performance of microfluidic capillary electrophoresis coupled to mass spectrometry (MCE-MS) as a rapid tool for profiling mAb biosimilar candidates from clonal cell lines. A representative originator sample was used to develop the MCE-MS method. The addition of dimethylsulfoxide (DMSO) to the background electrolyte yielded up to 60-fold enhancement of the protein MS signal. The resulting electropherograms consistently provided resolution of mAb charge variants within 10 min. Deconvoluted mass spectra facilitated the identification of basic variants such as C-terminal lysine and proline amidation, while the acidic variants could be assigned to deamidated forms. The MCE-MS method also allowed the identification of 18 different glycoforms in biosimilar samples. To mimic early-stage cell line selection, samples from five clonal cell lines that all expressed the same biosimilar candidate mAb were compared to their originator mAb. Based on the similarity observed in charge variants and glycoform profiles acquired by MCE-MS, the most promising candidate could be selected. The MCE-MS method demonstrated good overall reproducibility, as confirmed by a transferability study involving two separate laboratories. This study highlights the efficacy of the MCE-MS method for rapid proteoform screening of clonal cell line samples, underscoring its potential significance as an analytical tool in biosimilar process development., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Hydroxypropyl methyl cellulose (HPMC) is a type of cellulose derivative with properties that render it useful in e.g. food, cosmetics, and pharmaceutical industry. The substitution degree and composition of the β-glucose subunits of HPMC affect its physical and functional properties, but HPMC characterization is challenging due to its high structural heterogeneity, including many isomers. In this study, comprehensive two-dimensional liquid chromatography-mass spectrometry was used to examine substituted glucose monomers originating from complete acid hydrolysis of HPMC. Resolution between the different monomers was achieved using a C18 and cyano column in the first and second LC dimension, respectively. The data analysis process was structured to obtain fingerprints of the monomers of interest. The results revealed that isomers of the respective monomers could be selectively separated based on the position of substituents. The examination of two industrial HPMC products revealed differences in overall monomer composition. While both products contained monomers with a similar degree of substitution, they exhibited distinct regioselectivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has emerged as a tool to study protein conformational states. In TIMS, gas-phase ions are guided across the IM stages by applying direct current (DC) potentials (D1-6), which, however, might induce changes in protein structures through collisional activation. To define conditions for native protein analysis, we evaluated the influence of these DC potentials using the metalloenzyme bovine carbonic anhydrase (BCA) as primary test compound. The variation of DC potentials did not change BCA-ion charge and heme content but affected (relative) charge-state intensities and adduct retention. Constructed extracted-ion mobilograms and corresponding collisional cross-section (CCS) profiles gave useful insights in (alterations of) protein conformational state. For BCA, the D3 and D6 potential (which are applied between the deflection transfer and funnel 1 [F1] and the accumulation exit and the start of the ramp, respectively) had most profound effects, showing multimodal CCS distributions at higher potentials indicating gradual unfolding. The other DC potentials only marginally altered the CCS profiles of BCA. To allow for more general conclusions, five additional proteins of diverse molecular weight and conformational stability were analyzed, and for the main protein charge states, CCS profiles were constructed. Principal component analysis (PCA) of the obtained data showed that D1 and D3 exhibit the highest degree of correlation with the ratio of folded and unfolded protein (F/U) as extracted from the mobilograms obtained per set D potential. The correlation of D6 with F/U and protein charge were similar, and D2, D4, and D5 showed an inverse correlation with F/U but were correlated with protein charge. Although DC boundary values for induced conformational changes appeared protein dependent, a set of DC values could be determined, which assured native analysis of most proteins., (© 2024 The Authors. Journal of Mass Spectrometry published by John Wiley & Sons Ltd.)

Background: Therapeutic monoclonal antibodies (mAbs) comprise a large structural variability with respect to charge, size and post-translational modifications. These critical quality attributes (CQAs) need to be assessed during and after the production of mAbs. This normally requires off-line purification and sample preparation as well as several chromatographic selectivities, which makes the whole process time-consuming and error-prone. To improve on this, we developed an integrated and automated multi-dimensional analytical platform for the simultaneous assessment of multiple CQAs of mAbs in cell culture fluid (CCF) from upstream processes., Results: The on-line system allows mAb characterization at the intact level, combining protein A affinity chromatography (ProtA) with size-exclusion, ion-exchange, and reversed-phase liquid chromatographic modes with UV and mass spectrometric detection. Multiple heart cuts of a single mAb elution band from ProtA are stored in 20-μL loops and successively sent to the multimethod options in the second dimension. ProtA loading and elution conditions and their compatibility with second-dimension LC modes were studied and optimized. Subsequently, heart-cutting and valve-switching schemes were investigated to achieve effective and reproducible analyses. The applicability of the developed workflow was demonstrated by the direct analysis (i.e. not requiring off-line sample preparation) of a therapeutic mAb in CCF, obtaining useful information on accurate molecular mass, glycosylation, and charge and size variants of the mAb product at the same time and in just over 1 h., Significance: The developed multidimensional platform is the first system that allows for multiple fractions from a single ProtA band to be characterized using different chromatographic selectivities in a single run allowing direct correlation between CQAs. The performance of the system is comparable to established off-line methods, fully compatible with upstream process samples, and provides a significant time-reduction of the characterization procedure., Competing Interests: Declaration of competing interest The authors declare the following financial interests/ which may be considered as potential competing interests: Govert W. Somsen reports equipment was provided by Agilent Technologies Inc., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Analytical methods for the assessment of drug-delivery systems (DDSs) are commonly suitable for characterizing individual DDS properties, but do not allow determination of several properties simultaneously. A comprehensive online two-dimensional liquid chromatography (LC × LC) system was developed that is aimed to be capable of characterizing both nanoparticle size and encapsulated cargo over the particle size distribution of a DDS by using one integrated method. Polymeric nanoparticles (NPs) with encapsulated hydrophobic dyes were used as model DDSs. Hydrodynamic chromatography (HDC) was used in the first dimension to separate the intact NPs and to determine the particle size distribution. Fractions from the first dimension were taken comprehensively and disassembled online by the addition of an organic solvent, thereby releasing the encapsulated cargo. Reversed-phase liquid chromatography (RPLC) was used as a second dimension to separate the released dyes. Conditions were optimized to ensure the complete disassembly of the NPs and the dissolution of the dyes during the solvent modulation step. Subsequently, stationary-phase-assisted modulation (SPAM) was applied for trapping and preconcentration of the analytes, thereby minimizing the risk of analyte precipitation or breakthrough. The developed HDC × RPLC method allows for the characterization of encapsulated cargo as a function of intact nanoparticle size and shows potential for the analysis of API stability.

The time required for method development in gradient-elution liquid chromatography (LC) may be reduced by using an empirical modelling approach to describe and predict analyte retention and peak width. However, prediction accuracy is impaired by system-induced gradient deformation, which can be especially prominent for steep gradients. As the deformation is unique to each LC instrument, it needs to be corrected for if retention modelling for optimization and method transfer is to become generally applicable. Such a correction requires knowledge of the actual gradient profile. The latter has been measured using capacitively coupled "contactless" conductivity detection (C 4 D), featuring a low detection volume (approximately 0.05 μL) and compatibility with very high pressures (80 MPa or more). Several different solvent gradients, from water to acetonitrile, water to methanol, and acetonitrile to tetrahydrofuran, could be measured directly without the addition of a tracer component to the mobile phase, exemplifying the universal nature of the approach. Gradient profiles were found to be unique for each solvent combination, flowrate, and gradient duration. The profiles could be described by convoluting the programmed gradient with a weighted sum of two distribution functions. Knowledge of the exact profiles was used to improve the inter-system transferability of retention models for toluene, anthracene, phenol, emodin, sudan-I and several polystyrene standards., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Size-exclusion chromatography (SEC) employing aqueous mobile phases with volatile salts at neutral pH combined with native mass spectrometry (nMS) is a valuable tool to characterize proteins and protein aggregates in their native state. However, the liquid-phase conditions (high salt concentrations) frequently used in SEC-nMS hinder the analysis of labile protein complexes in the gas phase, necessitating higher desolvation-gas flow and source temperature, leading to protein fragmentation/dissociation. To overcome this issue, we investigated narrow SEC columns (1.0 mm internal diameter, I.D.) operated at 15-μL/min flow rates and their coupling to nMS for the characterization of proteins, protein complexes and higher-order structures (HOS). The reduced flow rate resulted in a significant increase in the protein-ionization efficiency, facilitating the detection of low-abundant impurities and HOS up to 230 kDa (i.e., the upper limit of the Orbitrap-MS instrument used). More-efficient solvent evaporation and lower desolvation energies allowed for softer ionization conditions (e.g., lower gas temperatures), ensuring little or no structural alterations of proteins and their HOS during transfer into the gas phase. Furthermore, ionization suppression by eluent salts was decreased, permitting the use of volatile-salt concentrations up to 400 mM. Band broadening and loss of resolution resulting from the introduction of injection volumes exceeding 3% of the column volume could be circumvented by incorporating an online trap-column containing a mixed-bed ion-exchange (IEX) material. The online IEX-based solid-phase extraction (SPE) or "trap-and-elute" set-up provided on-column focusing (sample preconcentration). This allowed the injection of large sample volumes on the 1-mm I.D. SEC column without compromising the separation. The enhanced sensitivity attained by the micro-flow SEC-MS, along with the on-column focusing achieved by the IEX precolumn, provided picogram detection limits for proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

New psychoactive substances (NPS) are synthetic derivatives of illicit drugs designed to mimic their psychoactive effects. NPS are typically not controlled under drug acts or their legal status depends on their molecular structure. Discriminating isomeric forms of NPS is therefore crucial for forensic laboratories. In this study, a trapped ion mobility spectrometry time-of-flight mass spectrometry (TIMS-TOFMS) approach was developed for the identification of ring-positional isomers of synthetic cathinones, a class of compounds representing two-third of all NPS seized in Europe in 2020. The optimized workflow features narrow ion-trapping regions, mobility calibration by internal reference, and a dedicated data-analysis tool, allowing for accurate relative ion-mobility assessment and high-confidence isomer identification. Ortho-, meta- and para-isomers of methylmethcathinone (MMC) and bicyclic ring isomers of methylone were assigned based on their specific ion mobilities within 5 min, including sample preparation and data analysis. The resolution of two distinct protomers per cathinone isomer added to the confidence in identification. The developed approach was successfully applied to the unambiguous assignment of MMC isomers in confiscated street samples. These findings demonstrate the potential of TIMS-TOFMS for forensic case work requiring fast and highly-confident assignment cathinone-drug isomers in confiscated samples., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, we present high-throughput (HT) venomics, a novel analytical strategy capable of performing a full proteomic analysis of a snake venom within 3 days. This methodology comprises a combination of RP-HPLC-nanofractionation analytics, mass spectrometry analysis, automated in-solution tryptic digestion, and high-throughput proteomics. In-house written scripts were developed to process all the obtained proteomics data by first compiling all Mascot search results for a single venom into a single Excel sheet. Then, a second script plots each of the identified toxins in so-called Protein Score Chromatograms (PSCs). For this, for each toxin, identified protein scores are plotted on the y -axis versus retention times of adjacent series of wells in which a toxin was fractionated on the x -axis. These PSCs allow correlation with parallel acquired intact toxin MS data. This same script integrates the PSC peaks from these chromatograms for semiquantitation purposes. This new HT venomics strategy was performed on venoms from diverse medically important biting species; Calloselasma rhodostoma , Echis ocellatus , Naja pallida , Bothrops asper , Bungarus multicinctus , Crotalus atrox , Daboia russelii , Naja naja , Naja nigricollis , Naja mossambica , and Ophiophagus hannah . Our data suggest that high-throughput venomics represents a valuable new analytical tool for increasing the throughput by which we can define venom variation and should greatly aid in the future development of new snakebite treatments by defining toxin composition.

We report an online analytical platform based on the coupling of asymmetrical flow field-flow fractionation (AF4) and native mass spectrometry (nMS) in parallel with UV-absorbance, multi-angle light scattering (MALS), and differential-refractive-index (UV-MALS-dRI) detectors to elucidate labile higher-order structures (HOS) of protein biotherapeutics. The technical aspects of coupling AF4 with nMS and the UV-MALS-dRI multi-detection system are discussed. The "slot-outlet" technique was used to reduce sample dilution and split the AF4 effluent between the MS and UV-MALS-dRI detectors. The stability, HOS, and dissociation pathways of the tetrameric biotherapeutic enzyme (anticancer agent) l-asparaginase (ASNase) were studied. ASNase is a 140 kDa homo-tetramer, but the presence of intact octamers and degradation products with lower molecular weights was indicated by AF4-MALS/nMS. Exposing ASNase to 10 mM NaOH disturbed the equilibrium between the different non-covalent species and led to HOS dissociation. Correlation of the information obtained by AF4-MALS (liquid phase) and AF4-nMS (gas phase) revealed the formation of monomeric, tetrameric, and pentameric species. High-resolution MS revealed deamidation of the main intact tetramer upon exposure of ASNase to high pH (NaOH and ammonium bicarbonate). The particular information retrieved from ASNase with the developed platform in a single run demonstrates that the newly developed platform can be highly useful for aggregation and stability studies of protein biopharmaceuticals.

The properties of a polymeric material are influenced by its underlying molecular distributions, including the molecular-weight (MWD), chemical-composition (CCD), and/or block-length (BLD) distributions. Gradient-elution liquid chromatography (LC) is commonly used to determine the CCD. Due to the limited solubility of polymers, samples are often dissolved in strong solvents. Upon injection of the sample, such solvents may lead to broadened or poorly shaped peaks and, in unfavourable cases, to "breakthrough" phenomena, where a part of the sample travels through the column unretained. To remedy this, a technique called size-exclusion-chromatography gradients or gradient size-exclusion chromatography (gSEC) was developed in 2011. In this work, we aim to further explore the potential of gSEC for the analysis of the CCD, also in comparison with conventional gradient-elution reversed-phase LC, which in this work corresponded to gradient-elution reversed-phase liquid chromatography (RPLC). The influence of the mobile-phase composition, the pore size of the stationary-phase particles, and the column temperature were investigated. The separation of five styrene/ethyl acrylate copolymers was studied with one-dimensional RPLC and gSEC. RPLC was shown to lead to a more-accurate CCD in shorter analysis time. The separation of five styrene/methyl methacrylate copolymers was also explored using comprehensive two-dimensional (2D) LC involving gSEC, i.e. SEC × gSEC and SEC × RPLC. In 2D-LC, the use of gSEC was especially advantageous as no breakthrough could occur., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Cellulose ethers (CEs) are semi-synthetic polymers produced by derivatization of natural cellulose, yielding highly substituted products such as ethyl hydroxyethyl cellulose (EHEC) or methyl ethyl hydroxyethyl cellulose (MEHEC). CEs are commonly applied as pharmaceutical excipients and thickening agents in paints and drymix mortars. CE properties, such as high viscosity in solution, solubility, and bio-stability are of high interest to achieve required product qualities, which may be strongly affected by the substitution pattern obtained after derivatization. The average and molar degree of substitution often cannot explain functional differences observed among CE batches, and more in-depth analysis is needed. In this work, a new method was developed for the comprehensive mapping of the substitution degree and composition of β-glucose monomers of CE samples. To this end, CEs were acid-hydrolyzed and then analyzed by gradient reversed-phase liquid chromatography-mass spectrometry (LC-MS) using an acid-stable LC column and time-of-flight (TOF) mass spectrometer. LC-MS provided monomer resolution based on ethylene oxide, hydroxyl, and terminating methyl/ethyl content, allowing the assignment of detailed compositional distributions. An essential further distinction of constitutional isomer distributions was achieved using an in-house developed probability-based deconvolution algorithm. Aided by differential heat maps for visualization and straightforward interpretation of the measured LC-MS data, compositional variation between bio-stable and non-bio-stable CEs could be identified using this new approach. Moreover, it disclosed unexpected methylations in EHEC samples. Overall, the obtained molecular information on relevant CE samples demonstrated the method's potential for the study of CE structure-property relationships., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The majority of liquid chromatography (LC) methods are still developed in a conventional manner, that is, by analysts who rely on their knowledge and experience to make method development decisions. In this work, a novel, open-source algorithm was developed for automated and interpretive method development of LC(-mass spectrometry) separations ("AutoLC"). A closed-loop workflow was constructed that interacted directly with the LC system and ran unsupervised in an automated fashion. To achieve this, several challenges related to peak tracking, retention modeling, the automated design of candidate gradient profiles, and the simulation of chromatograms were investigated. The algorithm was tested using two newly designed method development strategies. The first utilized retention modeling, whereas the second used a Bayesian-optimization machine learning approach. In both cases, the algorithm could arrive within 4-10 iterations ( i.e. , sets of method parameters) at an optimum of the objective function, which included resolution and analysis time as measures of performance. Retention modeling was found to be more efficient while depending on peak tracking, whereas Bayesian optimization was more flexible but limited in scalability. We have deliberately designed the algorithm to be modular to facilitate compatibility with previous and future work ( e.g. , previously published data handling algorithms).

Envenomation by elapid snakes primarily results in neurotoxic symptoms and, consequently, are the primary focus of therapeutic research concerning such venoms. However, mounting evidence suggests these venoms can additionally cause coagulopathic symptoms, as demonstrated by some Asian elapids and African spitting cobras. This study sought to investigate the coagulopathic potential of venoms from medically important elapids of the genera Naja (true cobras), Hemachatus (rinkhals), and Dendroaspis (mambas). Crude venoms were bioassayed for coagulant effects using a plasma coagulation assay before RPLC/MS was used to separate and identify venom toxins in parallel with a nanofractionation module. Subsequently, coagulation bioassays were performed on the nanofractionated toxins, along with in-solution tryptic digestion and proteomics analysis. These experiments were then repeated on both crude venoms and on the nanofractionated venom toxins with the addition of either the phospholipase A 2 (PLA 2 ) inhibitor varespladib or the snake venom metalloproteinase (SVMP) inhibitor marimastat. Our results demonstrate that various African elapid venoms have an anticoagulant effect, and that this activity is significantly reduced for cobra venoms by the addition of varespladib, though this inhibitor had no effect against anticoagulation caused by mamba venoms. Marimastat showed limited capacity to reduce anticoagulation in elapids, affecting only N. haje and H. haemachatus venom at higher doses. Proteomic analysis of nanofractionated toxins revealed that the anticoagulant toxins in cobra venoms were both acidic and basic PLA 2 s, while the causative toxins in mamba venoms remain uncertain. This implies that while PLA 2 inhibitors such as varespladib and metalloproteinase inhibitors such as marimastat are viable candidates for novel snakebite treatments, they are not likely to be effective against mamba envenomings.

Synthetic polymers typically show dispersity in molecular weight and potentially in chemical composition. For the analysis of the chemical-composition distribution (CCD) gradient liquid chromatography may be used. The CCD obtained using this method is often convoluted with an underlying molecular-weight distribution (MWD). In this paper we demonstrate that the influence of the MWD can be reduced using very steep gradients and that such gradients are best realized utilizing recycling gradient liquid chromatography (LC↻LC). This method allows for a more-accurate determination of the CCD and the assessment of (approximate) critical conditions (if these exist), even when high-molecular-weight standards of narrow dispersity are not readily available. The performance and usefulness of the approach is demonstrated for several polystyrene standards, and for the separation of statistical copolymers consisting of styrene/methyl methacrylate and methyl methacrylate/butyl methacrylate. For the latter case, approximate critical compositions of the copolymers were calculated from the critical compositions of two homopolymers and one copolymer of known chemical composition, allowing for a determination of the CCD of unknown samples. Using this approach it is shown that the copolymers elute significantly closer to the predicted critical compositions after recycling of the gradient. This is most clear for the lowest-molecular-weight copolymer (M w  = 4.2 kDa), for which the difference between measured and predicted elution composition decreases from 7.9% without recycling to 1.4% after recycling., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2022. Published by Elsevier B.V.)

In many areas, studying photostability or the mechanism of photodegradation is of high importance. Conventional methods to do so can be rather time-consuming, laborious, and prone to experimental errors. In this paper we evaluate an integrated and fully automated system for the study of light-induced degradation, comprising a liquid handler, an irradiation source and exposure cell with dedicated optics and spectrograph, and a liquid chromatography (LC) system. A liquid core waveguide (LCW) was used as an exposure cell, allowing efficient illumination of the sample over a 12 cm path length. This cell was coupled to a spectrograph, allowing in situ absorbance monitoring of the exposed sample during irradiation. The LCW is gas-permeable, permitting diffusion of air into the cell during light exposure. This unit was coupled online to LC with diode array detection for immediate and automated analysis of the composition of the light-exposed samples. The analytical performance of the new system was established by assessing linearity, limit of detection, and repeatability of the in-cell detection, sample recovery and carryover, and overall repeatability of light-induced degradation monitoring, using riboflavin as the test compound. The applicability of the system was demonstrated by recording a photodegradation time profile of riboflavin.

Alterations in protein structure may have profound effects on biological function. Analytical techniques that permit characterization of proteins while maintaining their conformational and functional state are crucial for studying changes in the higher order structure of proteins and for establishing structure-function relationships. Coupling of native protein separations with mass spectrometry is emerging rapidly as a powerful approach to study these aspects in a reliable, fast and straightforward way. This Review presents the available native separation modes for proteins, covers practical considerations on the hyphenation of these separations with mass spectrometry and highlights the involvement of affinity-based separations to simultaneously obtain structural and functional information of proteins. The impact of these approaches is emphasized by selected applications addressing biomedical and biopharmaceutical research questions., (© 2022. Springer Nature Limited.)

Asymmetrical flow field-flow fractionation (AF4) has attracted considerable attention as a size-based separation technique, due to its mild separation conditions, broad working range (from approximately 10 3 to 10 9  Da molecular mass or from 1 nm to 1 μm particle diameter), and versatility. AF4 is primarily being used to measure particle size, polydispersity, and physical stability of various systems, such as (bio)-macromolecules and nanoparticles. In comparison with size-exclusion chromatography (packed column), AF4 (open channel) allows separation while preserving labile structures. Monitoring of interactions between different compounds and in very complex matrices is possible. Preservation of the structure and correlation of structural characteristics with activity and functionality can bolster the development of new therapeutic strategies for diseases and new materials with improved properties. In this review, a detailed overview is presented of developments in AF4 for interaction studies between various systems, such as protein-protein, polymer-polymer, nanoparticle-drug, and nanoparticle-protein. The prospects and obstacles for AF4, and other less-commonly used types of FFF, for studying interactions within complex and fragile systems are covered. Coupling AF4 to a variety of detection systems can greatly contribute to the understanding of the interaction/association processes and provide information on the interaction kinetics. This review is intended to provide comprehensive documentation on the types of information (structural, morphological, chemical) on molecular interactions that can be retrieved by AF4., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

This chapter focuses on the application of capillary zone electrophoresis hyphenated with mass spectrometry (CZE-MS) for the characterization of monoclonal antibodies (mAbs). mAbs are complex molecules comprising different glycoforms and many other posttranslational modifications. In addition to this inherent microheterogeneity, misassembling of antibodies can take place during production contributing to their macroheterogeneity. CZE-MS is a versatile and powerful technique which has demonstrated high potential for the assessment of both micro- and macroheterogeneity of mAbs. In this chapter, technical and practical considerations for the characterization of mAbs by CZE-MS are described. CE-MS interfacing, capillary coatings for the prevention of mAb adsorption, and sample preparation considerations are covered in detail. The assessment of the macro- and microheterogeneity is discussed and exemplified through three different approaches involving analysis of intact, enzymatically digested, and reduced antibodies. The examples also illustrate the use of two commercially available interfacing techniques (i.e., sheath liquid and sheathless) as well as different types of capillary coatings (positively charged and neutral coatings)., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Trifluoroacetic acid (TFA) is commonly used as mobile phase additive to improve retention and peak shape characteristics in hydrophilic interaction liquid chromatography (HILIC) of intact proteins. However, when using electrospray ionization-mass spectrometry (ESI-MS) detection, TFA may cause ionization suppression and adduct formation, leading to reduced analyte sensitivity. To address this, we describe a membrane-based microfluidic chip with multiple parallel channels for the selective post-column removal of TFA anions from HILIC. An anion-exchange membrane was used to physically separate the column effluent from a stripper flow solution comprising acetonitrile, formic acid, and propionic acid. The exchange of ions allowed the post-column removal of TFA used during HILIC separation of model proteins. The multichannel design of the device allows the use of flow rates of 0.2 mL/min without the need for a flow splitter, using mobile phases containing 0.1% TFA (13 mM). Separation selectivity and efficiency were maintained (with minor band broadening effects) while increasing the signal intensity and peak areas by improving ionization and reducing TFA adduct formation.

Snakebite is classified as a priority Neglected Tropical Disease by the World Health Organization. Understanding the pathology of individual snake venom toxins is of great importance when developing more effective snakebite therapies. Snake venoms may induce a range of pathologies, including haemolytic activity. Although snake venom-induced erythrocyte lysis is not the primary cause of mortality, haemolytic activity can greatly debilitate victims and contributes to systemic haemotoxicity. Current assays designed for studying haemolytic activity are not suitable for rapid screening of large numbers of toxic compounds. Consequently, in this study, a high-throughput haemolytic assay was developed that allows profiling of erythrocyte lysis, and was validated using venom from a number of medically important snake species (Calloselasma rhodostoma, Daboia russelii, Naja mossambica, Naja nigricollis and Naja pallida). The assay was developed in a format enabling direct integration into nanofractionation analytics, which involves liquid chromatographic separation of venom followed by high-resolution fractionation and subsequent bioassaying (and optional proteomics analysis), and parallel mass spectrometric detection. Analysis of the five snake venoms via this nanofractionation approach involving haemolytic assaying provided venom-cytotoxicity profiles and enabled identification of the toxins responsible for haemolytic activity. Our results show that the elapid snake venoms (Naja spp.) contained both direct and indirect lytic toxins, while the viperid venoms (C. rhodostoma and D. russelii) only showed indirect lytic activities, which required the addition of phospholipids to exert cytotoxicity on erythrocytes. The haemolytic venom toxins identified were mainly phospholipase A 2 s and cytotoxic three finger toxins. Finally, the applicability of this new analytical method was demonstrated using a conventional snakebite antivenom treatment and a small-molecule drug candidate to assess neutralisation of venom cytotoxins., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: beta-lactoglobulin (BLG) is one of the major cow's milk proteins and the most abundant allergen in whey. Heating is a common technologic treatment applied during milk transformational processes. Maillardation of BLG in the presence of reducing sugars and elevated temperatures may influence its antigenicity and allergenicity., Primary Objective: to analyze and identify lactosylation sites by capillary electrophoresis mass spectrometry (CE-MS)., Secondary Objective: to assess the effect of lactosylated BLG on antigenicity and degranulation of mast cells., Methods: BLG was lactosylated at pH 7, a water activity (aw) of 0.43, and a temperature of 65 °C using a molar ratio BLG:lactose of 1:1 by incubating for 0, 3, 8, 16 or 24 h. For the determination of the effect on antibody-binding capacity of lactosylated BLG, an ELISA was performed. For the assessment of degranulation of the cell-line RBL-hεIa-2B12 transfected with the human α-chain, Fcε receptor type 1 (FcεRI) was used., Results: BLG showed saturated lactosylation between 8 and 16 incubation hours in our experimental setup. Initial stage lactosylation sites L1 (N-terminus)-K47, K60, K75, K77, K91, K138 and K141-have been identified using CE-MS. Lactosylated BLG showed a significant reduction of both the IgG binding ( p = 0.0001) as well as degranulation of anti-BLG IgE-sensitized RBL-hεIa-2B12 cells ( p < 0.0001)., Conclusions and Clinical Relevance: this study shows that lactosylation of BLG decreases both the antigenicity and degranulation of mast cells and can therefore be a promising approach for reducing allergenicity of cow's milk allergens provided that the process is well-controlled.

Trapped ion-mobility spectrometry combined with quadrupole time-of-flight mass spectrometry (TIMS-QTOFMS) was evaluated as a tool for resolving linear and branched isomeric polyester oligomers. Solutions of polyester samples were infused directly into the ion source employing electrospray ionization (ESI). TIMS-MS provides both mobility and m / z data on the formed ions, allowing construction of extracted-ion mobilograms (EIMs). EIMs of polyester molecules showed multimodal patterns, indicating conformational differences among isomers. Subsequent TIMS-MS/MS experiments indicated mobility differences to be caused by (degree of) branching. These assignments were supported by liquid chromatography-TIMS-MS/MS analysis, confirming that direct TIMS-MS provided fast (500 ms/scan) distinction between linear and branched small oligomers. Observing larger oligomers (up to 3000 Da) using TIMS required additional molecular charging to ensure ion entrapment within the mobility window. Molecular supercharging was achieved using m -nitrobenzyl alcohol (NBA). The additional charges on the oligomer structures enhanced mobility separation of isomeric species but also added to the complexity of the obtained fragmentation mass spectra. This complexity could be partly reduced by post-TIMS analyte-decharging applying collision-induced dissociation (CID) prior to Q1 with subsequent isolation of the singly charged ions for further fragmentation. The as-obtained EIM profiles were still quite complex as larger molecules possess more possible structural isomers. Nevertheless, distinguishing between linear and symmetrically branched oligomers was possible based on measured differences in collisional cross sections (CCSs). The established TIMS-QTOFMS approach reliably allows branching information on isomeric polyester molecules up to 3000 Da to be obtained in less than 1 min analysis time.

Many organic compounds undergo changes under the influence of light. This might be beneficial in, for example, water purification, but undesirable when cultural-heritage objects fade or when food ingredients (e.g., vitamins) degrade. It is often challenging to establish a strong link between photodegradation products and their parent molecules due to the complexity of the sample. To allow effective study of light-induced degradation (LID), a low-volume exposure cell was created in which solutes are efficiently illuminated (especially at low concentrations) while simultaneously analysed by absorbance spectroscopy. The new LID cell encompasses a gas-permeable liquid-core waveguide (LCW) connected to a spectrograph allowing collection of spectral data in real-time. The aim of the current study was to evaluate the overall performance of the LID cell by assessing its transmission characteristics, the absolute photon flux achieved in the LCW, and its capacity to study solute degradation in presence of oxygen. The potential of the LID set-up for light-exposure studies was successfully demonstrated by monitoring the degradation of the dyes eosin Y and crystal violet.

Bites from elapid snakes typically result in neurotoxic symptoms in snakebite victims. Neurotoxins are, therefore, often the focus of research relating to understanding the pathogenesis of elapid bites. However, recent evidence suggests that some elapid snake venoms contain anticoagulant toxins which may help neurotoxic components spread more rapidly. This study examines the effects of venom from the West African black-necked spitting cobra ( Naja nigricollis ) on blood coagulation and identifies potential coagulopathic toxins. An integrated RPLC-MS methodology, coupled with nanofractionation, was first used to separate venom components, followed by MS, proteomics and coagulopathic bioassays. Coagulation assays were performed on both crude and nanofractionated N. nigricollis venom toxins as well as PLA 2 s and 3FTx purified from the venom. Assays were then repeated with the addition of either the phospholipase A 2 inhibitor varespladib or the snake venom metalloproteinase inhibitor marimastat to assess whether either toxin inhibitor is capable of neutralizing coagulopathic venom activity. Subsequent proteomic analysis was performed on nanofractionated bioactive venom toxins using tryptic digestion followed by nanoLC-MS/MS measurements, which were then identified using Swiss-Prot and species-specific database searches. Varespladib, but not marimastat, was found to significantly reduce the anticoagulant activity of N. nigricollis venom and MS and proteomics analyses confirmed that the anticoagulant venom components mostly consisted of PLA 2 proteins. We, therefore, conclude that PLA 2 s are the most likely candidates responsible for anticoagulant effects stimulated by N. nigricollis venom.

Rapid optimization of gradient liquid chromatographic (LC) separations often utilizes analyte retention modelling to predict retention times as function of eluent composition. However, due to the dwell volume and technical imperfections, the actual gradient may deviate from the set gradient in a fashion unique to the employed instrument. This makes accurate retention modelling for gradient LC challenging, in particular when very fast separations are pursued. Although gradient deformation has been addressed in method-transfer situations, it is rarely taken into account when reporting analyte retention parameters obtained from gradient LC data, hampering the comparison of data from various sources. In this study, a response-function-based algorithm was developed to determine analyte retention parameters corrected for geometry-induced deformations by specific LC instruments. Out of a number of mathematical distributions investigated as response-functions, the so-called "stable function" was found to describe the formed gradient most accurately. The four parameters describing the model resemble the statistical moments of the distribution and are related to chromatographic parameters, such as dwell volume and flow rate. The instrument-specific response function can then be used to predict the actual shape of any other gradient programmed on that instrument. To incorporate the predicted gradient in the retention modelling of the analytes, the model was extended to facilitate an unlimited number of linear gradient steps to solve the equations numerically. The significance and impact of distinct gradient deformation for fast gradients was demonstrated using three different LC instruments. As a proof of principle, the algorithm and retention parameters obtained on a specific instrument were used to predict the retention times on different instruments. The relative error in the predicted retention times went down from an average of 9.8% and 12.2% on the two other instruments when using only a dwell-volume correction to 2.1% and 6.5%, respectively, when using the proposed algorithm. The corrected retention parameters are less dependent on geometry-induced instrument effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Protein dynamics play a significant role in many aspects of enzyme activity. Monitoring of structural changes and aggregation of biotechnological enzymes under native conditions is important to safeguard their properties and function. In this work, the potential of asymmetrical flow field-flow fractionation (AF4) to study the dynamic association equilibria of the enzyme β-D-galactosidase (β-D-Gal) was evaluated. Three commercial products of β-D-Gal were investigated using carrier liquids containing sodium chloride or ammonium acetate, and the effect of adding magnesium (II) chloride to the carrier liquid was assessed. Preservation of protein structural integrity during AF4 analysis was essential and the influence of several parameters, such as the focusing step (including use of frit-inlet), cross flow, and injected amount, was studied. Size-exclusion chromatography (SEC) and dynamic light scattering (DLS) were used to corroborate the in-solution enzyme oligomerization observed with AF4. In contrast to SEC, AF4 provided sufficiently mild separation conditions to monitor protein conformations without disturbing the dynamic association equilibria. AF4 analysis showed that ammonium acetate concentrations above 40 mM led to further association of the dimers ("tetramerization") of β-D-Gal. Magnesium ions, which are needed to activate β-D-Gal, appeared to induce dimer association, raising justifiable questions about the role of divalent metal ions in protein oligomerization and on whether tetramers or dimers are the most active form of β-D-Gal., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

This chapter aims to explore various parameters involved in achieving high-end capillary electrophoresis hyphenated to mass spectrometry (CE-MS) analysis of proteins, peptides, and their posttranslational modifications. The structure of the topics discussed in this book chapter is conveniently mapped on the scheme of the CE-MS system itself, starting from sample preconcentration and injection techniques and finishing with mass analyzer considerations. After going through the technical considerations, a variety of relevant applications for this analytical approach are presented, including posttranslational modifications analysis, clinical biomarker discovery, and its growing use in the biotechnological industry., (© 2021. Springer Nature Switzerland AG.)

Repurposing small molecule drugs and drug candidates is considered as a promising approach to revolutionise the treatment of snakebite envenoming. In this study, we investigated the inhibiting effects of the small molecules varespladib (nonspecific phospholipase A 2 inhibitor), marimastat (broad spectrum matrix metalloprotease inhibitor) and dimercaprol (metal ion chelator) against coagulopathic toxins found in Crotalinae (pit vipers) snake venoms. Venoms from Bothrops asper , Bothrops jararaca , Calloselasma rhodostoma and Deinagkistrodon acutus were separated by liquid chromatography, followed by nanofractionation and mass spectrometry identification undertaken in parallel. Nanofractions of the venom toxins were then subjected to a high-throughput coagulation assay in the presence of different concentrations of the small molecules under study. Anticoagulant venom toxins were mostly identified as phospholipases A 2 , while procoagulant venom activities were mainly associated with snake venom metalloproteinases and snake venom serine proteases. Varespladib was found to effectively inhibit most anticoagulant venom effects, and also showed some inhibition against procoagulant toxins. Contrastingly, marimastat and dimercaprol were both effective inhibitors of procoagulant venom activities but showed little inhibitory capability against anticoagulant toxins. The information obtained from this study aids our understanding of the mechanisms of action of toxin inhibitor drug candidates, and highlights their potential as future snakebite treatments., (© 2020 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

A single-step copolymerization strategy was developed for the preparation of carbohydrate (glucose and maltose) functionalized monoliths using click reaction. Firstly, novel carbohydrate-functionalized methacrylate monomers were synthesized through Cu(I)-catalyzed 1,3-dipolar cycloaddition (alkyne-azide reaction) of terminal alkyne with azide of carbohydrate derivatives. The corresponding carbohydrate functionalized monolithic columns were then prepared through a single-step in-situ copolymerization. The physicochemical properties and performance of the fabricated monolithic columns were evaluated using scanning electron microscopy, Fourier-transform infrared spectroscopy, and nano-liquid chromatography. For the optimized monolithic column, satisfactory column permeability and good separation performance were demonstrated for polar compounds including nucleoside, phenolic compounds and benzoic acid derivatives. The monolithic column is also highly useful for selective and efficient enrichment of glycopeptides from human IgG tryptic digests. This study not only provided a novel hydrophilic column for separation and selective trapping of polar compounds, but also proposed a facile and efficient approach for preparing carbohydrate functionalized monoliths., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Many organisms, ranging from plants to mammals, contain phospholipase A 2 enzymes (PLA 2 s), which catalyze the production of lysophospholipids and fatty acid proinflammatory mediators. PLA 2 s are also common constituents of animal venoms, including bees, scorpions and snakes, and they cause a wide variety of toxic effects including neuro-, myo-, cyto-, and cardio-toxicity, anticoagulation and edema. The aim of this study was to develop a generic method for profiling enzymatically active PLA 2 s in snake venoms after chromatographic separation. For this, low-volume high-throughput assays for assessment of enzymatic PLA 2 activity were evaluated and optimized. Subsequently, the assays were incorporated into a nanofractionation platform that combines high-resolution fractionation of crude venoms by liquid chromatography (LC) with bioassaying in 384-well plate format, and parallel mass spectrometric (MS) detection for toxin identification. The miniaturized assays developed are based on absorbance or fluorescence detection (respectively, using cresol red or fluorescein as pH indicators) to monitor the pH drop associated with free fatty acid formation by enzymatically active PLA 2 s. The methodology was demonstrated for assessment of PLA 2 activity profiles of venoms from the snake species Bothrops asper, Echis carinatus, Echis coloratus, Echis ocellatus, Oxyuranus scutellatus and Daboia russelii russelii., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Current approaches to study glycosylation of polyclonal human immunoglobulins G (IgG) usually imply protein digestion or glycan release. While these approaches allow in-depth characterization, they also result in a loss of valuable information regarding certain subclasses, allotypes and co-occuring post-translational modifications (PTMs). Unfortunately, the high variability of polyclonal IgGs makes their intact mass spectrometry (MS) analysis extremely challenging. We propose here a middle-up strategy for the analysis of the intact fragment crystallizable (Fc) region of human plasma IgGs, with the aim of acquiring integrated information of the N -glycosylation and other PTMs of subclasses and allotypes. Human plasma IgG was isolated using Fc-specific beads followed by an on-bead C H 2 domain digestion with the enzyme IdeS. The obtained mixture of Fc subunits was analyzed by capillary electrophoresis (CE) and hydrophilic interaction liquid chromatography (HILIC) hyphenated with MS. CE-MS provided separation of different IgG-subclasses and allotypes, while HILIC-MS allowed resolution of the different glycoforms and their oxidized variants. The orthogonality of these techniques was key to reliably assign Fc allotypes. Five individual donors were analyzed using this approach. Heterozygosis was observed in all the analyzed donors resulting in a total of 12 allotypes identified. The assignments were further confirmed using recombinant monoclonal IgG allotypes as standards. While the glycosylation patterns were similar within allotypes of the same subclass, clear differences were observed between IgG subclasses and donors, highlighting the relevance of the proposed approach. In a single analysis, glycosylation levels specific for each allotype, relative abundances of subclasses and information on co-occurring modifications are obtained. This middle-up method represents an important step toward a comprehensive analysis of immunoglobulin G-Fc variants., (Copyright © 2020 Sénard, Gargano, Falck, de Taeye, Rispens, Vidarsson, Wuhrer, Somsen and Domínguez-Vega.)

Animal-derived antivenoms are the only specific therapies currently available for the treatment of snake envenoming, but these products have a number of limitations associated with their efficacy, safety and affordability for use in tropical snakebite victims. Small molecule drugs and drug candidates are regarded as promising alternatives for filling the critical therapeutic gap between snake envenoming and effective treatment. In this study, by using an advanced analytical technique that combines chromatography, mass spectrometry and bioassaying, we investigated the effect of several small molecule inhibitors that target phospholipase A 2 (varespladib) and snake venom metalloproteinase (marimastat, dimercaprol and DMPS) toxin families on inhibiting the activities of coagulopathic toxins found in Viperinae snake venoms. The venoms of Echis carinatus , Echis ocellatus , Daboia russelii and Bitis arietans , which are known for their potent haemotoxicities, were fractionated in high resolution onto 384-well plates using liquid chromatography followed by coagulopathic bioassaying of the obtained fractions. Bioassay activities were correlated to parallel recorded mass spectrometric and proteomics data to assign the venom toxins responsible for coagulopathic activity and assess which of these toxins could be neutralized by the inhibitors under investigation. Our results showed that the phospholipase A 2 -inhibitor varespladib neutralized the vast majority of anticoagulation activities found across all of the tested snake venoms. Of the snake venom metalloproteinase inhibitors, marimastat demonstrated impressive neutralization of the procoagulation activities detected in all of the tested venoms, whereas dimercaprol and DMPS could only partially neutralize these activities at the doses tested. Our results provide additional support for the concept that combinations of small molecules, particularly the combination of varespladib with marimastat, serve as a drug-repurposing opportunity to develop new broad-spectrum inhibitor-based therapies for snakebite envenoming.

Phospholipase A 2 (PLA 2 ) enzymes are important toxins found in many snake venoms, and they can exhibit a variety of toxic activities including causing hemolysis and/or anticoagulation. In this study, the inhibiting effects of the small molecule PLA 2 inhibitor varespladib on snake venom PLA 2 s was investigated by nanofractionation analytics, which combined chromatography, mass spectrometry (MS), and bioassays. The venoms of the medically important snake species Bothrops asper , Calloselasma rhodostoma , Deinagkistrodon acutus , Daboia russelii , Echis carinatus , Echis ocellatus, and Oxyuranus scutellatus were separated by liquid chromatography (LC) followed by nanofractionation and interrogation of the fractions by a coagulation assay and a PLA 2 assay. Next, we assessed the ability of varespladib to inhibit the activity of enzymatic PLA 2 s and the coagulopathic toxicities induced by fractionated snake venom toxins, and identified these bioactive venom toxins and those inhibited by varespladib by using parallel recorded LC-MS data and proteomics analysis. We demonstrated here that varespladib was not only capable of inhibiting the PLA 2 activities of hemotoxic snake venoms, but can also effectively neutralize the coagulopathic toxicities (most profoundly anticoagulation) induced by venom toxins. While varespladib effectively inhibited PLA 2 toxins responsible for anticoagulant effects, we also found some evidence that this inhibitory molecule can partially abrogate procoagulant venom effects caused by different toxin families. These findings further emphasize the potential clinical utility of varespladib in mitigating the toxic effects of certain snakebites.

Many industrial enzymes exhibit macro- and micro-heterogeneity due to co-occurring post-translational modifications. The resulting proteoforms may have different activity and stability and, therefore, the characterization of their distributions is of interest in the development and monitoring of enzyme products. Protein glycosylation may play a critical role as it can influence the expression, physical and biochemical properties of an enzyme. We report the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) to profile intact glycoform distributions of high mannose-type N-glycosylated proteins, using an industrially produced fungal lipase for the food industry as an example. We compared these results with conventional reversed phase LC-MS (RPLC-MS) and sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE). HILIC appeared superior in resolving lipase heterogeneity, facilitating mass assignment of N-glycoforms and sequence variants. In order to understand the glycoform selectivity provided by HILIC, fractions from the four main HILIC elution bands for lipase were taken and subjected to SDS-PAGE and bottom-up proteomic analysis. These analyses enabled the identification of the most abundant glycosylation sites present in each fraction and corroborated the capacity of HILIC to separate protein glycoforms based on the number of glycosylation sites occupied. Compared to RPLC-MS, HILIC-MS reducted the sample complexity delivered to the mass spectrometer, facilitating the assignment of the masses of glycoforms and sequence variants as well as increasing the number of glycoforms detected (69 more proteoforms, 177% increase). The HILIC-MS method required relatively short analysis time (<30 min), in which over 100 glycoforms were distinguished. We suggest that HILIC(-MS) can be a valuable tool in characterizing bioengineering processes aimed at steering protein glycoform expression as well as to check the consistency of product batches., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The proliferation of increasingly more sophisticated analytical separation systems, often incorporating increasingly more powerful detection techniques, such as high-resolution mass spectrometry, causes an urgent need for highly efficient data-analysis and optimization strategies. This is especially true for comprehensive two-dimensional chromatography applied to the separation of very complex samples. In this contribution, the requirement for chemometric tools is explained and the latest developments in approaches for (pre-)processing and analyzing data arising from one- and two-dimensional chromatography systems are reviewed. The final part of this review focuses on the application of chemometrics for method development and optimization., (© 2020 The Authors. Journal of Separation Science published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Snakebites cause upwards of 1.8 million envenomings, 138,000 deaths and 500,000 cases of long term morbidity each year. Viper snake venoms (family Viperidae) generally contain a high proportion of proteases which can cause devastating effects such as hemorrhage, coagulopathy, edema, necrosis, and severe pain, in envenomed victims. In this study, analytical techniques were combined with enzymatic assays to develop a novel method for the detection of snake venom protease activity by using rhodamine-110-peptide substrate. In the so called at-line nanofractionation set up, crude venoms were first separated with reversed phase liquid chromatography, after which fractions were collected onto 384-well plates. Protease activity assays were then performed in the 384-well plates and bioassay chromatograms were constructed revealing protease activity. Parallel obtained UV absorbance, MS and proteomics data from a previous study facilitated toxin identification. The application of the rhodamine-110-peptide substrate assay showed significantly greater sensitivity compared to prior assays using casein-FITC as the substrate. Moreover, cross referencing UV and MS data and resulted in the detection of a number of tentative proteases suspected to exhibit protease activity, including snake venom serine proteases from Calloselasma rhodostoma and Daboia russelli venom and a snake venom metalloproteinase from the venom of Echis ocellatus. Our data demonstrate that his methodology can be a useful tool for selectively identifying snake venom proteases, and can be applied to provide a better understanding of protease-induced pathologies and the development of novel therapeutics for treating snakebite., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Snakebite is a neglected tropical disease that results in a variety of systemic and local pathologies in envenomed victims and is responsible for around 138,000 deaths every year. Many snake venoms cause severe coagulopathy that makes victims vulnerable to suffering life-threating haemorrhage. The mechanisms of action of coagulopathic snake venom toxins are diverse and can result in both anticoagulant and procoagulant effects. However, because snake venoms consist of a mixture of numerous protein and peptide components, high throughput characterizations of specific target bioactives is challenging. In this study, we applied a combination of analytical and pharmacological methods to identify snake venom toxins from a wide diversity of snake species that perturb coagulation. To do so, we used a high-throughput screening approach consisting of a miniaturised plasma coagulation assay in combination with a venom nanofractionation approach. Twenty snake venoms were first separated using reversed-phase liquid chromatography, and a post-column split allowed a small fraction to be analyzed with mass spectrometry, while the larger fraction was collected and dispensed onto 384-well plates. After fraction collection, any solvent present in the wells was removed by means of freeze-drying, after which it was possible to perform a plasma coagulation assay in order to detect coagulopathic activity. Our results demonstrate that many snake venoms simultaneously contain both procoagulant and anticoagulant bioactives that contribute to coagulopathy. In-depth identification analysis from seven medically-important venoms, via mass spectrometry and nanoLC-MS/MS, revealed that phospholipase A2 toxins are frequently identified in anticoagulant venom fractions, while serine protease and metalloproteinase toxins are often associated with procoagulant bioactivities. The nanofractionation and proteomics approach applied herein seems likely to be a valuable tool for the rational development of next-generation snakebite treatments by facilitating the rapid identification and fractionation of coagulopathic toxins, thereby enabling specific targeting of these toxins by new therapeutics such as monoclonal antibodies and small molecule inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Size-exclusion chromatography employing aqueous mobile phases with volatile salts at neutral pH combined with electrospray-ionization mass spectrometry (SEC-ESI-MS) is a useful tool to study proteins in their native state. However, whether the applied eluent conditions actually prevent protein-stationary phase interactions, and/or protein denaturation, often is not assessed. In this study, the effects of volatile mobile phase additives on SEC retention and ESI of proteins were thoroughly investigated. Myoglobin was used as the main model protein, and eluents of varying ionic strength and pH were applied. The degree of interaction between protein and stationary phase was evaluated by calculating the SEC distribution coefficient. Protein-ion charge state distributions obtained during offline and online native ESI-MS were used to monitor alterations in protein structure. Interestingly, most of the supposedly mild eluent compositions induced nonideal SEC behavior and/or protein unfolding. SEC experiments revealed that the nature, ionic strength, and pH of the eluent affected protein retention. Protein-stationary phase interactions were effectively avoided using ammonium acetate at ionic strengths above 0.1 M. Direct-infusion ESI-MS showed that the tested volatile eluent salts seem to follow the Hofmeister series: no denaturation was induced using ammonium acetate (kosmotropic), whereas ammonium formate and bicarbonate (both chaotropic) caused structural changes. Using a mobile phase of 0.2 M ammonium acetate (pH 6.9), several proteins (i.e., myoglobin, carbonic anhydrase, and cytochrome c) could be analyzed by SEC-ESI-MS using different column chemistries without compromising their native state. Overall, with SEC-ESI-MS, the effect of nonspecific interactions between protein and stationary phase on the protein structure can be studied, even revealing gradual structural differences along a peak.

Venomous snakebite is one of the world's most lethal neglected tropical diseases. Animal-derived antivenoms are the only standardized specific therapies currently available for treating snakebite envenoming, but due to venom variation, often this treatment is not effective in counteracting all clinical symptoms caused by the multitude of injected toxins. In this study, the coagulopathic toxicities of venoms from the medically relevant snake species Bothrops asper , Calloselasma rhodostoma , Deinagkistrodon acutus , Daboia russeli i , Echis carinatus and Echis ocellatus were assessed. The venoms were separated by liquid chromatography (LC) followed by nanofractionation and parallel mass spectrometry (MS). A recently developed high-throughput coagulation assay was employed to assess both the pro- and anticoagulant activity of separated venom toxins. The neutralization capacity of antivenoms on separated venom components was assessed and the coagulopathic venom peptides and enzymes that were either neutralized or remained active in the presence of antivenom were identified by correlating bioassay results with the MS data and with off-line generated proteomics data. The results showed that most snake venoms analyzed contained both procoagulants and anticoagulants. Most anticoagulants were identified as phospholipases A 2 s (PLA 2 s) and most procoagulants correlated with snake venom metalloproteinases (SVMPs) and serine proteases (SVSPs). This information can be used to better understand antivenom neutralization and can aid in the development of next-generation antivenom treatments., Competing Interests: The authors declare no conflict of interest

This chapter introduces bioactivity and bioaffinity terms in relation to mixture profiling and gives the significance of bioactivity and/or bioaffinity profiling of biologically active mixtures in general, and for bioactive mixtures in drug discovery research in particular. Further, the chapter gives an overview of the common and less common analytical approaches for bioactivity profiling of bioactive mixtures. Special focus is put on bioassay-guided fractionation as the standard technique employed (in identification and purification of bioactive molecules from a bioactive mixture), and on state-of-the-art post-column bioactivity profiling approaches, also providing examples and limitations of these analytical methods. On-column and pre-column bioactivity profiling analytics is also discussed. Examples of bioactive molecules identified and purified from different natural products are given with emphasis on molecules isolated from animal venoms. Finally, this chapter briefly discusses the importance of bioactivity profiling of metabolic mixtures in drug discovery.

We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1-30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator. The influence of the solenoid supply voltage on solvent release was determined and the effect of the frequency, flow rate, and mobile phase composition was studied. For this purpose, droplet release was visually assessed for a wide range of frequencies and flow rates, followed by quantitative evaluation of a selection of promising settings for highly accurate, repeatable, and stable fraction collection. The potential of the new fraction collector for LC-based bioactivity screening was demonstrated by fractionating the LC eluent of a mixture of estrogenic and androgenic compounds, and a surface water sample (blank and spiked with bioactives) combining mass spectrometric detection and two reporter gene assays for bioactivity detection of the fractions. Additionally, a mixture of two compounds was repeatedly LC separated and fractionated to assess the feasibility of the system for analyte isolation followed by nuclear magnetic resonance analysis.

A CZE method was validated and implemented for fast and accurate in-process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus vector-based vaccines. An analytical-quality-by-design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.

Protein glycosylation is one of the most common and critical post-translational modification, which results from covalent attachment of carbohydrates to protein backbones. Glycosylation affects the physicochemical properties of proteins and potentially their function. Therefore it is important to establish analytical methods which can resolve glycoforms of glycoproteins. Recently, hydrophilic-interaction liquid chromatography (HILIC)-mass spectrometry has demonstrated to be a useful tool for the efficient separation and characterization of intact protein glycoforms. In particular, amide-based stationary phases in combination with acetonitrile-water gradients containing ion-pairing agents, have been used for the characterization of glycoproteins. However, finding the optimum gradient conditions for glycoform resolution can be quite tedious as shallow gradients (small decrease of acetonitrile percentage in the elution solvent over a long time) are required. In the present study, the retention mechanism and peak capacity of HILIC for non-glycosylated and glycosylated proteins were investigated and compared to reversed-phase liquid chromatography (RPLC). For both LC modes, ln k vs. φ plots of a series of test proteins were calculated using linear solvent strength (LSS) analysis. For RPLC, the plots were spread over a wider φ range than for HILIC, suggesting that HILIC methods require shallower gradients to resolve intact proteins. Next, the usefulness of computer-aided method development for the optimization of the separation of intact glycoform by HILIC was examined. Five retention models including LSS, adsorption, and mixed-mode, were tested to describe and predict glycoprotein retention under gradient conditions. The adsorption model appeared most suited and was applied to the gradient prediction for the separation of the glycoforms of six glycoproteins (Ides-digested trastuzumab, alpha-acid glycoprotein, ovalbumin, fetuin and thyroglobulin) employing the program PIOTR. Based on the results of three scouting gradients, conditions for high-efficiency separations of protein glycoforms varying in the degree and complexity of glycosylation was achieved, thereby significantly reducing the time needed for method optimization., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A novel analytical method based on hybrid trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has been developed to achieve fast enantiomeric separation of amino acids (AAs). Resolution of chiral AAs was achieved by forming diastereomers through derivatization with the chiral agent (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), avoiding the use of reference compounds. Electrospray ionization (ESI) in positive mode yielded sodiated FLEC-AAs ions of which the diastereomers could be separated by TIMS. The effect of other alkali metal ions (such as Li and K) on the enantioselectivity was studied, but chiral discrimination was only observed for Na. TIMS conditions, including voltage ramp, ramp time, and accumulation time were optimized for each AA, and collision cross sections (CCSs) were determined for all diastereomers. The migration order of the DL enantiomers was found to be dependent on the structure of the AA. The resulting TIMS resolution (K0/ΔK0) for the FLEC-AA diastereomers on average was 115, requiring a mobility (K0) difference of about 0.009 cm 2 /(V s) to achieve 50%-valley separation. From the 21 AAs studied, enantiomer separation was achieved for 17 AAs with mobility differences ranging from 0.009 for lysine up to 0.061 cm 2 /(V s) for asparagine. Moreover, the presented methodology provided mutual separation of various AAs, allowing chiral analysis of multiple AAs simultaneously which may be challenging with previous enantioselective IMS approaches. It appeared possible to fully resolve all studied DL-AAs using three distinct TIMS methods, resulting in a total MS run time of about 3 min (1 min per method) and a total analysis time (including derivatization) of less than 15 min. The method demonstrated capable to determine enantiomeric ratios down to 2.5% with detection limits for the D enantiomers in the nanomolar range. This new TIMS-based methodology opens up possibilities for easy and fast analysis of AA enantiomers.

Natural extracts are complex mixtures that may be rich in useful bioactive compounds and therefore are attractive sources for new leads in drug discovery. This review describes drug discovery from natural products and in explaining this process puts the focus on ion-channel drug discovery. In particular, the identification of bioactives from natural products targeting nicotinic acetylcholine receptors (nAChRs) and serotonin type 3 receptors (5-HT 3 Rs) is discussed. The review is divided into three parts: "Targets," "Sources," and "Approaches." The "Targets" part will discuss the importance of ion-channel drug targets in general, and the α7-nAChR and 5-HT 3 Rs in particular. The "Sources" part will discuss the relevance for drug discovery of finding bioactive compounds from various natural sources such as venoms and plant extracts. The "Approaches" part will give an overview of classical and new analytical approaches that are used for the identification of new bioactive compounds with the focus on targeting ion channels. In addition, a selected overview is given of traditional venom-based drug discovery approaches and of diverse hyphenated analytical systems used for screening complex bioactive mixtures including venoms.

In the field of metabolomics, CE-MS is now recognized as a strong analytical technique for the analysis of (highly) polar and charged metabolites in a wide range of biological samples. Over the past few years, significant attention has been paid to the design and improvement of CE-MS approaches for (large-scale) metabolic profiling studies and for establishing protocols in order to further expand the role of CE-MS in metabolomics. In this paper, which is a follow-up of a previous review paper covering the years 2014-2016 (Electrophoresis 2017, 38, 190-202), main advances in CE-MS approaches for metabolomics studies are outlined covering the literature from July 2016 to June 2018. Aspects like developments in interfacing designs and data analysis tools for improving the performance of CE-MS for metabolomics are discussed. Representative examples highlight the utility of CE-MS in the fields of biomedical, clinical, microbial, and plant metabolomics. A complete overview of recent CE-MS-based metabolomics studies is given in a table, which provides information on sample type and pretreatment, capillary coatings and MS detection mode. Finally, some general conclusions and perspectives are given., (© 2018 The Authors. Electrophoresis Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)