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Abstract: We present a label-free biosensor capable to map local potential by photogenerating charge carriers in a field effect device. It consists in an amplitude-modulated laser beam scanning through an electrolyte/insulator/semiconductor structure at very low illumination around the equilibrium state of semiconductor. A proof of principle is demonstrated by depositing a self-assembled monolayer on the dielectric and by showing a clear photopotential change between naked and covered areas. [Copyright &y& Elsevier]

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 DOWEX TM resin. However, experimental results allowed us to demonstrate that the presence of DOWEX TM resin colloid residues in the aqueous phase greatly improves the quality of the spectra obtained by MALDI-TOFMS on a Fondaparinux model glycosaminoglycan.

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 AA 2A R, which were confirmed by competition experiments and whose K d values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

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 AA 2A R used as a model protein, which was not possible before. The affinity was confirmed by competition experiments.

Glycosaminoglycans (GAGS) are involved in many biological processes through interactions with a variety of proteins, including proteases, growth factors, cytokines, chemokines and adhesion molecules. Identifying druggable GAG-protein interactions for therapeutic purposes is a challenge for the analytical community. In this context, this work investigates the use of a new miniaturized monolithic affinity column (poly(GMA-co-MBA) grafted with antithrombin III (AT III)) to specifically capture and elute high affinity sequences contained in low molecular weight heparin (enoxaparin) for further on-line characterization. This miniaturized, high binding capacity affinity column allows the specific capture of high-affinity oligosaccharide chains from Enoxaparin, even at low concentrations and with a minimal consumption of AT III. In addition to purification, this elution process enables preconcentration for direct analysis by capillary zone electrophoresis. It was found that many of oligosaccharide chains in enoxaparin were eliminated and that certain chain sequences were retained and enriched. Direct coupling with MALDI-TOF MS was successfully used to further characterize the specifically retained oligosaccharides where nano-ESI-TOF MS failed. After optimization of the sample preparation and ionization parameters, direct on-line analysis was performed by applying the elution volume released from the miniaturized affinity column (≤1 μL) directly to the MALDI plate. Finally, this original miniaturized analytical workflow coupling miniaturized AT III-affinity chromatography to MALDI-TOF MS detection is able to select, enrich and detect and identify high affinity sequences (mainly DP4 in size length with a high degree of sulfation) from low molecular weight heparin samples. A more specific selection of GAG sequences can be achieved by increasing the ionic strength during the washing step of affinity chromatography. This is consistent with the known binding pattern between heparin and AT III., 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 Elsevier B.V. All rights reserved.)

We report an original methodology based on affinity chromatography coupled with mass spectrometry to decipher the complexity of dynamic combinatorial libraries (DCLs) of glycoclusters. Such libraries are intended to boost the design of potential therapeutic anti-infectious agents targeting Pseudomonas aeruginosa, which is responsible for numerous diseases, mostly found in hospitals as major a cause of nosocomial infections. Dynamic combinatorial chemistry provides a rapid access to an equilibrating mixture of glycocluster candidates through the formation of reversible covalent bonds under thermodynamic control. Identifying each molecule in the complex mixture overcomes challenges due to the dynamic process. Selection of glycoclusters candidates was first realized on a model lectin (Concanavalin A, ConA). Home-made affinity nanocolumns, containing covalently immobilized ConA and have volumes in the microliter range, were used to separate DCLs of glycoclusters with respect to their specific lectin binding properties under buffered aqueous conditions. Miniaturization facilitates the inline coupling with MS detection in such purely aqueous and buffered conditions and reduces target protein consumption. Monolithic lectin-affinity columns prepared by immobilization of ConA were first characterized using a known ligand. The amount of active binding immobilized lectin is 61 ± 5 pmol on 8.5-cm length column. We demonstrated the ability of our approach to evaluate individual dissociation constants of species directly in the complex mixture. The concept was then successfully applied to the screening of DCLs of more complex glycoclusters to identify (by mass spectrometry) and rank the ligands (by relative breakthrough curve delay) according to their affinity for the immobilized lectin in a single experiment., 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 Elsevier B.V. All rights reserved.)

In affinity chromatography, non-specific interactions between the ligands and the affinity column may affect the results, leading to misinterpretations during the investigation of protein-ligand interactions (detection of false positives in ligand screening, lack of specificity in purification). Such non-specific interactions may arise both from the underlying support or from the target protein itself. If the second ones are protein-dependent (and cannot be studied in a general framework), the first ones occur in the same way regardless of the immobilized target. We propose a methodology to identify the origin of such non-specific interactions with the underlying material of the affinity column. This methodology relies on the systematic investigation of the retention behavior of a set of 41 low-molecular weight compounds covering a wide chemical space (net charge, log D, functionality). We first demonstrate that the main source of non-specific interactions on the most commonly used GMA-co-EDMA monolith comes from hydrophobic effects. To reduce such non-specific interactions, we developed a new hydrophilic glycidyl methacrylate-based monolith by replacing the EDMA crosslinker by the more hydrophilic NN' Methylenebisacrylamide (MBA). Optimization of the synthesis parameters (monomer content, initiation type, temperature) has focused on the reduction of non-specific interaction with the monolithic support while maximizing the amount of protein that can be grafted onto the monolith at the issue of its synthesis. The retention data of the 41 test solutes on the new poly(GMA-co-MBA) monolith shows a drastic reduction of non-specific interactions except for cationic compounds. The particular behavior of cationic compounds is due to their electrostatic interactions with carboxylic groups resulting from the partial acidic hydrolysis of amide groups of MBA during the epoxide ring opening step. So, the ring opening step in acidic media was replaced by a hot water treatment to avoid side reaction on MBA. The new monolith poly(GMA-co-MBA) not only has improved hydrophilic surface properties but also a higher protein density (16 ± 0.8 pmol cm -1 instead of 8 ± 0.3 pmol cm -1 ). To highlight the benefits of this new hydrophilic monolith for affinity chromatographic studies, frontal affinity chromatography experiments were conducted on these monoliths grafted with con A., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022. Published by Elsevier B.V.)

This work explores the capability of antithrombin III-functionalized capillary monolithic columns (in-line coupled with MS detection) to selectively capture, release and detect high affinity binders of antithrombin III (AT III) from oligosaccharides mixtures. The in-situ characterization of home-made AT III affinity columns was done by frontal affinity chromatography coupled to MS detection using fondaparinux as model ligand. Three different preparation methods of miniaturized antithrombin III monolithic affinity columns were optimized and compared. Immobilization of antithrombin III onto Concanavalin A functionalized column is the simplest method but leads to lowest protein density. The two other methods, direct grafting on aldehyde preactivated monoliths and immobilization of biotinylated antithrombin III to streptavidin-functionalized columns, require the presence of fondaparinux to protect the heparin binding site during the grafting process. Up to 1.3 ± 0.3 pmol cm -1 of antithrombin III were immobilized with both methods. The direct coupling of such miniaturized affinity columns to MS-detection was made possible by optimization of the elution step. Ammonia (0.1 M) was chosen as an efficient and MS compatible solvent to elute fondaparinux. Finally, the complete analytical workflow (capture/elution/detection) was demonstrated to allow the selective capture and elution of fondaparinux initially contained in a complex oligosaccharide mixture with a limit of detection of 1 pmol., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Frontal affinity chromatography is a powerful, underappreciated technique for the qualitative (screening) and quantitative ( K d determination) evaluation of biological interactions. Its development has been previously hampered by its sample consumption, limited throughput, and lack of dedicated instrumentation especially at a miniaturized scale. This work describes two original experimental devices allowing nano-frontal affinity chromatography titrations (nano-FAC) to be automatically implemented in the time-saving staircase mode. The first nano-FAC system utilizes a capillary electrophoresis device (7100 CE Agilent system) in the pressurization mode with in situ UV detection. The second nano-FAC experimental setup implements a nano-LC device (Ultimate 3000 Thermo) modified with a 10-port valve equipped with two superloops (loop volume, 5 μL) operating alternatively and automatically in a single run. The benefits and drawbacks of each approach are exemplified using two model protein-ligand interactions (concanavalin A-mannose and concanavalin A-glucose). The two methods result in concordant dissociation constants ( K d ) and number of active site ( B act ) values, obtained in a fully automated manner, with low sample consumption and good throughput.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interest or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier B.V.)

Susceptibility to autoimmune diabetes is a complex genetic trait. Linkage analyses exploiting the NOD mouse, which spontaneously develops autoimmune diabetes, have proved to be a useful tool for the characterization of some of these traits. In a linkage analysis using 3A9 TCR transgenic mice on both B10.BR and NOD.H2 k backgrounds, we previously determined that both the Idd2 and Idd13 loci were linked to the proportion of immunoregulatory CD4 - CD8 - double negative (DN) T cells. In addition to Idd2 and Idd13, five other loci showed weak linkage to the proportion of DN T cells. Of interest, in an interim analysis, a locus on chromosome 12 is linked to DN T cell proportion in both the spleen and the lymph nodes. To determine the impact of this locus on DN T cells, we generated two congenic sublines, which we named Chr12P and Chr12D for proximal and distal, respectively. While 3A9 TCR:insHEL NOD.H2 k -Chr12D mice were protected from diabetes, 3A9 TCR:insHEL NOD.H2 k -Chr12P showed an increase in diabetes incidence. Yet, the proportion of DN T cells was similar to the parental 3A9 TCR NOD.H2 k strain for both of these congenic sublines. A genome-wide two dimensional LOD score analysis reveals genetic epistasis between chromosome 12 and the Idd13 locus. Altogether, this study identified further complex genetic interactions in defining the proportion of DN T cells, along with evidence of genetic epistasis within a locus on chromosome 12 influencing autoimmune susceptibility.

Biophysical techniques that enable the screening and identification of weak affinity fragments against a target protein are at the heart of Fragment Based Drug Design approaches. In the case of membrane proteins, the crucial criteria for fragment screening are low protein consumption, unbiased conformational states and rapidity because of the difficulties in obtaining sufficient amounts of stable and functionally folded proteins. Here we show for the first time that lipid-nanodisc systems (membrane-mimicking environment) and miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach was exemplified using the AA 2A R GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA 2A R (only about 1 μg of protein per column) were fully characterized by frontal chromatographic experiments. This method allows (i) to distinguish specific and unspecific ligand/receptor interactions, (ii) to assess dissociation constants, (iii) to identify the binding pocket of uncharacterized ligands using a reference compound (whose binding site is known) with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range can be detected. At last, the applicability of this method was demonstrated with 6 fragments recently identified as ligands or non-ligands of AA 2A R., 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.)

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., Competing Interests: Declaration of Competing Interest Authors declare no conflict of interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)