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The study of biomolecules and their interactions in their natural environment requires increasingly sophisticated technological and methodological developments. The complexity of these developments is due, among other things, to the nature of these molecules and the small quantities available depending on their origin. In this context, this study focuses on the conditions for improving the detection of glycosaminoglycans on a miniaturized scale by mass spectrometry. These multicharged anionic linear polysaccharides are in fact difficult to study by mass spectrometry and can present, for a given molecule, a large number of signals linked to different charge states, to the loss of one or more sulfate groups and to the presence of different adducts, which reduces sensitivity and complicates the interpretation of the spectra. In order to reduce this complexity, we have investigated different sample preparation methods applicable to small sample volumes. The development of home-made capillary ion-exchange columns, for example, makes it possible to control the adducts formed in nano-ESI coupling. However, their use on a miniaturized scale for detection by MALDI-TOF-MS does not allow for performances as high as those obtained with treatment with a commercial DOWEXTM resin. However, experimental results allowed us to demonstrate that the presence of DOWEXTM resin colloid residues in the aqueous phase greatly improves the quality of the spectra obtained by MALDI-TOFMS on a Fondaparinux model glycosaminoglycan. [ABSTRACT FROM AUTHOR]

Following the rapid developments in genomics and proteomics in recent decades, the analysis of the associated sugars became a critical center of interest, particularly in the field of analytical chemistry. Indeed, their synthetic route, diversity and complexity make these carbohydrates, and in particular glycosaminoglycans (GAG), a real analytical puzzle. These polymers are involved in a wide range of biological processes through their particular interaction with proteins. This article aims to show the contribution of separation sciences to the identification and characterization of interaction sites on GAGs. After a brief review of the structure and diversity of GAGs, the question was: do GAGs have a specific interaction motif with proteins? A literature review showed that this question is still controversial and that the reality is more nuanced. This review presents the different separative strategies used to try to identify such a specific GAG binding site if there is one. [ABSTRACT FROM AUTHOR]

Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement. [ABSTRACT FROM AUTHOR]

The identification of weak-affinity ligands targeting membrane proteins is of great interest in Fragment-Based Drug Design (FBDD). Recently, miniaturized weak affinity chromatography (WAC) has been proposed as a valuable tool to study interactions between small ligands and wild-type membrane proteins embedded in so-called nanodisc biomimetic membranes immobilized on GMA-co-EDMA monoliths in situ-synthesized in capillary columns (less than one microliter in volume). In this proof-of-concept study, the achievable affinity range was limited to medium affinity (low micromolar range). The present work investigates different strategies to extend the affinity range towards low affinities, either by increasing the density of membrane proteins on the chromatographic support or by reducing non-specific interactions with the monolith. The combination of the use of a new and more hydrophilic monolithic support (poly(DHPMA-co-MBA)) and a multilayer nanodisc grafting process (up to three layers) allows a significant increase in the membrane protein density by a more than three-fold factor (up to 5.4 pmol cm−1). Such an increase in protein density associated with reduced non-specific interactions makes it possible to extend the range of detectable affinity, as demonstrated by the identification and characterization of affinities of very low-affinity ligands (Kd values of several hundred micromolar) for the adenosine receptor AA2AR used as a model protein, which was not possible before. The affinity was confirmed by competition experiments. [ABSTRACT FROM AUTHOR]

For complex sample analysis, separation of analytes from interfering compounds is mandatory for accurate quantification; this is the main goal of chromatography. 4Cause-and-effect relation map for isocratic separation in reverse-phase chromatography Fundamental laws and models In the set of relations reported in Figs. New maps will be drawn according to the targeted teaching goal, and we have also developed specific maps to address band broadening (from column and external effects), gas chromatography, capillary electrophoresis... How can we take this map a step further?. [Extracted from the article]

In miniaturized affinity chromatography, the development of hydrophilic organic monoliths with reduced non-specific interactions and high-protein grafting capacity remains a hot topic. In this work, we propose the one-step synthesis of a diol organic monolith to replace the gold-standard epoxy-based organic monoliths (which require post-modification, namely hydrolysis, prior to use). The synthesis of this new monolith builds upon the use of N-N'-Methylenebis(acrylamide) (MBA), as a hydrophilic crosslinker, and 2,3-dihydroxypropyl methacrylate (DHPMA), a diol monomer that eliminates the time-consuming epoxy ring opening step and its associated side reactions. The optimization of one-step synthesis parameters led to a monolith with a satisfactory permeability ((4.8 ± 0.5) × 10−14 m2), high efficiency (117,600 plates/m at optimum flow velocity (uopt = 0.09 cm s−1)) and reduced non-specific interactions. It is exemplified by its separation ability in the HILIC mode (separation of nucleosides), and by the retention data set of 41 test solutes, which were used to evaluate the non-specific interactions. This new poly(DHPMA-co-MBA) monolith has not only hydrophilic surface properties, but also improved protein grafting capacity compared to the glycidyl-based monolith (13 ± 0.7 pmol cm−1). The potential of this monolith is illustrated in affinity chromatography, where the concanavalin ligands are ranked according to their Kd values. [ABSTRACT FROM AUTHOR]

Affinity chromatography is an often overlooked chromatographic technique regarding receptor/ligand studies. Indeed, biologists have a tendency to focus on more recent biophysical techniques such as NMR or surface plasmon resonance methods, among others. However, in order to face challenges related to the use of membrane proteins in drug discovery, alternative and/or complementary methods are welcome. In this frame, chromatographic methods, through the affinity mode, bring new perspectives due to their relative simplicity and the great diversity of information that they can provide. Affinity chromatography methods rely on the immobilization of targets on the stationary phase and this step is crucial when dealing with membrane proteins. Different immobilization strategies with their own specificities (surface density, nonspecific interaction, complex surface chemistry of the taking or release step) were developed allowing to encompass a wide range of applications: from ligand-ranking (synthetic compounds of medium to high affinity) to fragment screening (small compounds of weak affinity), adsorption, distribution, metabolism, and excretion studies or identification of active components from natural extracts. [ABSTRACT FROM AUTHOR]

Boronate affinity chromatography (BAC) is commonly used in sample preparation techniques for its specific selectivity for cis-diol-containing compounds. Various chromatographic materials based on phenyl-boronic acid (PBA) functionalization have been designed. This review aims to present the versatility of the underlying BAC mechanisms. The PBA moiety presents secondary/complementary/interfering interactions, i.e., hydrophobic, electrostatic and/or hydrogen-bonding interactions. The comprehensive understandings of these mechanisms are very helpful to manipulate separation selectivity, which is a key feature in biomolecular sample treatment. Each BAC interaction is reviewed and illustrated by specific examples. Special attention is given to the underlying role of PBA supports for sugars, glycans and glycoconjugates recognition. [ABSTRACT FROM AUTHOR]

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This work focuses on the synthesis and characterization of porous silica monolith-based adsorbent materials. Materials with bimodal porosity (macro and meso) were prepared through a sol–gel process. The capacity of adsorption of organic molecules was ensured by grafting of hydrophobic organic coating on the silica surface. Alkylsilane chains or lauryl acrylate polymer were used for this purpose. The adsorption kinetic behavior of the produced materials was assessed through benzophenone adsorption studies in aqueous media. The results have shown that the macropore size of the monoliths had no effect on the adsorption capacity. The thicker organic layer prepared by polymerization of lauryl acrylate has decreased the adsorption kinetics without affecting the total adsorption capacity. The over-coating with additional external layer of hydrogel further slowed the diffusion of benzophenone thus better matches the passive-sampler requirements. [ABSTRACT FROM AUTHOR]

A new vinyltrimethoxysilane-based hybrid silica monolith was developed and used as a reversed-phase capillary column. The synthesis of this rich vinyl hybrid macroporous monolith, by cocondensation of vinyltrimethoxysilane with tetramethoxysilane, was investigated using an unconventional (formamide, nitric acid) porogen/catalyst system. A macroporous hybrid silica monolith with 80% in mass of vinyltrimethoxysilane in the feeding silane solution was obtained and compared to a more conventional low vinyl content hybrid monolith with only of 20% vinyltrimethoxysilane. Monoliths were characterized by scanning electron microscopy, 29Si nuclear magnetic resonance spectroscopy and N2 adsorption-desorption. About 80% of the vinyl precursor was incorporated in the final materials, leading to 15.9 and 61.5% of Si atoms bonded to vinyl groups for 20% vinyltrimethoxysilane and 80% vinyltrimethoxysilane, respectively. The 80% vinyltrimethoxysilane monolith presents a lower surface area than 20% vinyltrimethoxysilane (159 versus 551 m2/g), which is nevertheless compensated by a higher vinyl surface density. Chromatographic properties were evaluated in reversed-phase mode. Plots of ln( k) versus percentage of organic modifier were used to assess the reversed-phase mechanism. Its high content of organic groups leads to high retention properties. Column efficiencies of 170 000 plates/m were measured for this 80% vinyltrimethoxysilane hybrid silica monolith. Long capillary monolithic columns (90 cm) were successfully synthesized ( N = 120 000). [ABSTRACT FROM AUTHOR]

Trends in LC focus on dedicated separation developments spanning different fields of applications ranging from sample preparation to miniaturization. Chromatographic performances result from the porous media, its implantation inside the 'column,' and its surface functionalization. Because molecular interactions govern chromatographic phenomena, surface functionalization is still a hot research topic. Besides standard approaches for surface functionalization, the use of new surface chemistry reactions opens new perspectives. Click chemistry belongs to this new class of chemical reactions, characterized by its specificity, compatibility with aqueous media, and high reaction yields. In this frame, we review the use of click chemistry reactions in chromatographic sciences. In a first part, we present click chemistry with a specific focus on its implementation in stationary phases. The use of these new clicked materials is detailed and discussed with respect to the chromatographic mode. [ABSTRACT FROM AUTHOR]

In this work, we developed a surface functionalization way of silica monoliths with a rapid, simple, versatile, and localizable photografting step. The elaboration of a photoreactive layer at the surface of monoliths was first optimized. The functionalization with [γ-(methacryloyloxy)propyl]trimethoxysilane at 80°C in a hydro-organic solution containing triethylamine as catalyst allows reachng the highest density of methacrylate photoactive moieties on silica surfaces. These methacrylate reactive surfaces were subsequently photografted within few minutes with acrylate monomers bearing alkyl chains ( C12 and C18). The photografting efficiency was determined by monitoring the retentive properties of monoliths in the RP mode. The retention factors are of the same order of magnitude as highly retentive columns obtained by modification of silica surface with long-alkyl chain silanes or by thermal polymerization of long-alkyl chain monomers. It was also verified that such grafting neither impaired the efficiency of the monolithic stationary phase ( Hmin = 6-8 μm in nano- LC) nor its permeability (about 6 × 10−14 m2). Further, it was also demonstrated that photografting is localizable in nonmasked defined areas. Results obtained in anion-exchange chromatography after photopolymerization of [2-(methacryloyloxy)ethyl]trimethylammonium chloride are presented as well to demonstrate the versatility of the developed approach. [ABSTRACT FROM AUTHOR]

Dans cet article, l’auteur étudie la façon dont se sont comportés les Européens lors de la crise irakienne et, dans ce contexte, cherche à cerner les contours d’une éventuelle identité européenne. En 2002/2003, le conflit a agi comme un révélateur et a laissé apparaître d’importantes convergences dans la manière dont les opinions publiques européennes ont appréhendé la question de l’intervention militaire, le rapport aux Etats‐Unis et le rôle de l’Union européenne sur la scène internationale. On ne peut certes avancer que les Européens se caractérisent par une culture diplomatique et stratégique commune, d’une part parce que les élites politiques ont pris des positions opposées, d’autre part parce que la période étudiée est trop brève pour en juger. Cependant, si l’on se réfère à un sentiment minimal d’appartenance commun et au partage de critères d’interprétation identiques, il semble qu’on puisse identifier en Europe les éléments d’une sphère publique en voie d’émergence. In this article the author examines the Europeans’ behaviour during the Iraq crisis and attempts to sketch European identity in this context. The conflict of 2002/2003 revealed how European public opinion apprehended the question of military intervention, relations to the United States and the role of the European Union on an international scale. It certainly can not be affirmed that Europeans are united in diplomatic and strategic policies, as, on the one hand, the political elite was at odds with itself and as, on the other, too short a period of time was analyzed in the article to be conclusive on this issue. However, elements of an emerging European public sphere can be discerned when referring to a minimal spirit of togetherness and to the sharing of identical criteria of interpretation. [ABSTRACT FROM AUTHOR]

In leathers, formaldehyde is currently analyzed according to EN ISO 17226-1 standard, by reversed phase liquid chromatography after off-line precolumn derivatization with 2,4 dinitrophenylhydrazine (DNPH) in strong acidic conditions. We first demonstrate that this standard is not adapted to leather retanned with resins likely to release formaldehyde by hydrolysis. Indeed, formaldehyde content may be largely overestimated due to concomitant resin hydrolysis (in harsh acidic conditions) that releases formaldehyde during the derivatization step and during the waiting time on autosampler before analysis. Therefore, we thoroughly studied the derivatization step in order to propose new derivatization conditions. Replacing orthophosphoric acid by less acidic buffer solutions is not enough to avoid hydrolysis. A derivatization without adding acid is realized by solubilizing DNPH in acetonitrile instead of orthophosphoric acid. These conditions lead to a complete derivatization of formaldehyde in 3 h at 50 °C (in a water bath) while avoiding the hydrolysis of co-extracted dicyandiamide and melamine resins. The as-obtained leather extracts are stable over time. Formaldehyde contents found with this method agree with the formaldehyde content measured immediately at the end of derivatization reaction in standard conditions or with formaldehyde content measured by a home-designed flow injection analysis with acetylacetone online derivatization and UV detection. [ABSTRACT FROM AUTHOR]

• Photoclick chemistry was used to prepare boronate affinity monolithic. • Stable and dense layer-like phenyl-bonorate layers were obtained. • In-line coupling of miniaturized boronate affinity monolith to RP-monolithic column. • Proof of feasibility of the in-line coupling was successfully demonstrated. In-line coupling of capillary columns is an effective means for achieving miniaturized and automated separation methods. The use of multimodal column designed to allow the direct integration of a sample preparation step to the separation column is one example. Herein we propose a novel in-line coupling at the capillary scale between a boronate affinity capillary column (μBAMC unit) and a reversed-phase separation column. This has been made possible due to the elaboration of a new and efficient μBAMC unit. A thiol-activated silica monolithic capillary column was functionalized through thiol-ene photoclick reaction. This simple and fast reaction allows to prepare stable μBAMC units having grafting densities of 1.93 ± 0.17 nmol cm−1. This grafting strategy increases the surface density by a factor 4 compared to our previous strategies and opens the frame to in-line coupling with reversed-phase capillary column. Proof of concept of the in-line coupling was done by coupling a 1-cm length μBAMC unit to a 7-cm length reversed phase capillary column. The conditions of loading, elution and separation were optimized for cis-diol nucleosides analysis (uridine, cytidine, adenosine, guanosine). A loading volume (at pH 8.5) of up to 21 hold volume (i.e 1 μl) of the μBAMC unit can be loaded without sample breakthrough. For the least retained nucleoside (uridine) a limit of detection of 50 ng mL−1 was estimated. Elution and full separation of the four nucleosides was triggered by flushing the multimodal column with an acetic acid (50 mM) / methanol (98/2, v/v) mobile phase. [ABSTRACT FROM AUTHOR]

• Biotin-streptavidin grafting approach is enhanced by Multi-layering. • Multilayering allows a 2-fold increase in biotinylated target grafting density. • Weak affinity nano-LC lower limit of affinity detection extended to 1mM Kd. Affinity chromatography is a powerful technique to identify and quantify weak ligand-protein interactions (Kd in the range of mM to 0.1µM). In some fields such as Fragment Based Drug Discovery, the detection of very weak affinities (mM) is of utmost importance since weak ligands can be good starting points for the conception of high affinity ligands. However, the identification of such weak ligands can be hampered by the limited bulk density of active target grafted onto the support. At the same time, downscaling the chromatographic column is of utmost interest when scarce and/or expensive proteins are targeted. In this context, we herein present a novel approach of protein immobilization to improve the bulk density of active protein grafted onto organic capillary monolithic columns. The proposed approach is based on the streptavidin-biotin interaction and consists of successive grafting steps of biotinylated target protein onto streptavidin layers through a multi-layering process. Concanavalin A was used as model protein. The study focuses on the optimization of the grafting conditions to maximize the amount of active protein during the multi-layering process and highlights the impact of the biotinylation ratio of the protein. It is demonstrated that a 3-layer grafting process allows to improve the bulk density of active sites by a 2-fold factor compared to a single layer. This improvement in protein density allows to increase the affinity range of this technique to the millimolar range. [ABSTRACT FROM AUTHOR]

Highlights • Comparison of organic and silica monolithic affinity capillary columns. • used with a capillary electrophoresis instrument and UV detection. • with a fast and efficient streptavidin-biotin monolith bonding. • and needing μg amounts of protein for interaction studies. Abstract Affinity monolith columns of 375 nL (effective length 8.5 cm, internal diameter 75 μm) were developed for protein-ligand affinity investigations needing only 3 μg of human serum albumin (HSA). To promote specific interactions and avoid non-specific ones, different combinations of monolithic supports and bio-functionalization pathways were evaluated. Silica and glycidylmethacrylate based monoliths were in-situ synthesized and grafted with HSA. Two direct grafting methods epoxy-amine and Schiff Base plus the streptavidin-biotin method were compared. The columns were evaluated by frontal analysis with ligands of known affinity for HSA. It is shown that a classical capillary electrophoresis instrument equipped with an external pressure device can be used to do weak affinity chromatography at low pressure (less than 1.2 MPa) in a fully automated way and with very low reagent consumption. The grafting pathways were compared in terms of (i) total and active amounts of immobilized protein, (ii) non-specific interactions, (iii) protein denaturation. According to these criteria, the organic monoliths combined with the streptavidin-biotin approach provided the best results. This immobilization pathway led to the highest active protein content (40 pmol of HSA per 8.5-cm column) with less than 10% non-specific interactions and 84% protein activity. The target grafting step lasts only 10 min and is UV-monitored, the UV breakthrough curve giving the exact amount of bound protein. This novel approach was validated by K d measurements of 3 known ligands of HSA. Streptavidin generic monolith columns could be stored at 4 °C for 3 months maintaining activity. μg of a biotin modified sensitive protein could be attached to a stable streptavidin monolith for immediate interaction studies avoiding stability problems. This development was subsequently extended to another protein of higher pharmaceutical interest: the N-terminal domain of HSP90. Affinity was measured for two known ligands and determined K d values were in accordance with the literature, proving that our technique is applicable to other proteins. [ABSTRACT FROM AUTHOR]

• An ITP method was optimized for the separation of two CS disaccharide isomers. • A miniaturized fractionation and collection of pure solutes was demonstrated. • By off-line characterization of the collected fractions by CZE and IRMPD spectroscopy. Glycans analysis is challenging due to their immense structural diversity. Isotachophoresis was investigated as separation method for the purification of isobaric sulfated disaccharides prior to their characterization by Mass Spectrometry (MS) and tunable IR multiple photon dissociation (IRMPD). This proof of feasibility study was applied to the separation and characterization of chondroitin sulfate (CS) disaccharides. ITP separation conditions were optimized. Separation starts using a 2.5 mM chloride ions and 10 mM glycine at pH 3.2 solution as leading electrolyte and a terminating electrolyte composed of formic acid 2.5 mM and glycine 10 mM at pH 3.5. The CS disaccharides sample were prepared in the terminating electrolyte. The length of injection was also investigated in order to create longer plateau-like bands of pure solutes. This strategy was helpful for collecting fraction at such microseparation scale. Indeed, capillary ITP affords the injection of few tens of nanoliter of sample. Fractionation of the CS disaccharides mixture in isolated ITP bands and collection of solutes were successfully done using a HPC coated fused silica capillary of 1m-length and 75 µm of internal diameter. Collected fractions in a final of volume 10 µL were analyzed by CZE, tandem MS and IRMPD spectroscopy. The purity of each fraction is higher than 90% and is well-adapted to IRMPD characterization. [ABSTRACT FROM AUTHOR]