Monolith weak affinity chromatography for μg-protein-ligand interaction study

Lecas, Lucile; Randon, Jérôme; Berthod, Alain; Dugas, Vincent; Demesmay, Claire

doi:10.1016/j.jpba.2019.01.012

Cited by 18 publications

(26 citation statements)

References 27 publications

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“…The most frequently used organic monoliths in AMC are based on copolymers of glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EDMA) [19,44,50,61–65]. GMA/EDMA monoliths can be readily synthesized and are commercially available under the trade name “convective interaction media” (CIM) [15,20,44].…”

Section: Supports For Amcmentioning

confidence: 99%

Affinity monolith chromatography: A review of general principles and recent developments

2021

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Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target‐specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal‐ion affinity chromatography, and dye‐ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.

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Section: Supports For Amcmentioning

confidence: 99%

Affinity monolith chromatography: A review of general principles and recent developments

2021

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“…Streptavidin columns were prepared using the Schiff base method as previously described 37 (Streptavidin from streptomyces avidinii, affinity purified, ≥ 13 U.mg -1 of protein, Sigma Aldrich)…”

Section: Streptavidin Immobilization Protocolmentioning

confidence: 99%

“…At this level of miniaturization, an insitu synthesized poly(GMA-co-EDMA) monolith chromatographic support was preferred to conventional packed particulate ones. The epoxy surface groups of the monolith were then hydrolyzed into diols that were subsequently oxidized into aldehyde capable of reacting with streptavidin through Schiff base reaction 37 . The resulting streptavidin-coated columns were finally used to immobilize the biotinylated nanodiscs containing AA2AR.…”

Section: Aa2ar Affinity Columns: Preparation and Evaluation With Known Ligandsmentioning

confidence: 99%

Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors

Lecas

Hartmann

Caro

et al. 2020

Analytica Chimica Acta

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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 lipidnanodisc systems (membrane-mimicking environment) and ultra-miniaturized affinity chromatography can be combined to identify specific small molecule ligands that bind to an integral membrane protein. The approach is exemplified using the AA2AR GPCR. Home-made affinity nano-columns modified with nanodiscs-embedded AA2AR (only about 1 µg of protein per column) are 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 with competition experiments. Weak affinity ligands with Kd in the low to high micromolar range be detected. At last, the applicability of this method is demonstrated with 6 fragments recently identified as ligands or non-ligands of AA2AR.

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“…Terms such as weak affinity chromatography (WAC) (Meiby et al, 2013; Singh P. et al, 2017; Ohlson and Duong-Thi, 2018; Lecas et al, 2019), high-performance affinity chromatography (HPAC) (Hage, 2017; Li Z. et al, 2017; Beeram et al, 2018; Zhang C. et al, 2018), high-performance liquid affinity chromatography (HPLAC) (Zheng et al, 2014), biointeraction chromatography (Wainer, 2004), affinity monolith chromatography (AMC) (Lecas et al, 2019), and cellular chromatography (CC) (Ciesla et al, 2016; Xu et al, 2019) have been largely and correctly employed, but all of them could be well-settled in the general term bioaffinity chromatography, which has been introduced to differentiate them from the classic affinity chromatography (de Moraes et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

“…Frontal bioaffinity chromatography has been employed in thermodynamic and kinetic analyses and can simultaneously provide information about the amount of immobilized protein, the number of active binding sites, and the equilibrium constants of these sites (Lecas et al, 2019). Additionally, the molecular interaction can be identified and characterized in a concentration-independent manner, and, in a mixture, it can be revealed in the range from millimolar to picomolar dissociation constants (Schriemer, 2008; Calleri et al, 2009; Temporini et al, 2013; Hage, 2017).…”

Section: Introductionmentioning

confidence: 99%

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

2019

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Ligand-target interactions play a central role in drug discovery processes because these interactions are crucial in biological systems. Small molecules-proteins interactions can regulate and modulate protein function and activity through conformational changes. Therefore, bioanalytical tools to screen new ligands have focused mainly on probing ligand-target interactions. These interactions have been evaluated by using solid-supported proteins, which provide advantages like increased protein stability and easier protein extraction from the reaction medium, which enables protein reuse. In some specific approaches, precisely in the ligand fishing assay, the bioanalytical method allows the ligands to be directly isolated from complex mixtures, including combinatorial libraries and natural products extracts without prior purification or fractionation steps. Most of these screening assays are based on liquid chromatography separation, and the binding events can be monitored through on-line or off-line methods. In the on-line approaches, solid supports containing the immobilized biological target are used as chromatographic columns most of the time. Several terms have been used to refer to such approaches, such as weak affinity chromatography, high-performance affinity chromatography, on-flow activity assays, and high-performance liquid affinity chromatography. On the other hand, in the off-line approaches, the binding event occurs outside the liquid chromatography system and may encompass affinity and activity-based assays in which the biological target is immobilized on magnetic particles or monolithic silica, among others. After the incubation step, the supernatant or the eluate from the binding assay is analyzed by liquid chromatography coupled to various detectors. Regardless of the selected bioanalytical approach, the use of solid supported proteins has significantly contributed to the development of automated and reliable screening methods that enable ligands to be isolated and characterized in complex matrixes without purification, thereby reducing costs and avoiding time-laborious steps. This review provides a critical overview of recently developed assays.

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Monolith weak affinity chromatography for μg-protein-ligand interaction study

Cited by 18 publications

References 27 publications

Affinity monolith chromatography: A review of general principles and recent developments

Affinity monolith chromatography: A review of general principles and recent developments

Miniaturized weak affinity chromatography for ligand identification of nanodiscs-embedded G-protein coupled receptors

Solid-Supported Proteins in the Liquid Chromatography Domain to Probe Ligand-Target Interactions

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