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Affinity monolith chromatography: a review of principles and recent analytical applications
Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis, or study of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments and applications of this method, with particular emphasis being given to work that has appeared in the last 5 years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths, and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns, and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal ions, and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized-metal-ion affinity chromatography, dye–ligand affinity chromatography, chiral separations, and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing, and biotechnology. Current trends and possible directions in AMC are also discussed.
Journal Article
Immobilized metal ion affinity chromatography: a review on its applications
After 35 years of development, immobilized metal ion affinity chromatography (IMAC) has evolved into a popular protein purification technique. This review starts with a discussion of its mechanism and advantages. It continues with its applications which include the purification of histidine-tagged proteins, natural metal-binding proteins, and antibodies. IMAC used in conjunction with mass spectroscopy for phosphoprotein fractionation and proteomics is also covered. Finally, this review addresses the developments, limitations, and considerations of IMAC in the biopharmaceutical industry.
Journal Article
Combined proximity labeling and affinity purification−mass spectrometry workflow for mapping and visualizing protein interaction networks
Affinity purification coupled with mass spectrometry (AP–MS) and proximity-dependent biotinylation identification (BioID) methods have made substantial contributions to interaction proteomics studies. Whereas AP−MS results in the identification of proteins that are in a stable complex, BioID labels and identifies proteins that are in close proximity to the bait, resulting in overlapping yet distinct protein identifications. Integration of AP–MS and BioID data has been shown to comprehensively characterize a protein’s molecular context, but interactome analysis using both methods in parallel is still labor and resource intense with respect to cell line generation and protein purification. Therefore, we developed the Multiple Approaches Combined (MAC)-tag workflow, which allows for both AP–MS and BioID analysis with a single construct and with almost identical protein purification and mass spectrometry (MS) identification procedures. We have applied the MAC-tag workflow to a selection of subcellular markers to provide a global view of the cellular protein interactome landscape. This localization database is accessible via our online platform (
http://proteomics.fi
) to predict the cellular localization of a protein of interest (POI) depending on its identified interactors. In this protocol, we present the detailed three-stage procedure for the MAC-tag workflow: (1) cell line generation for the MAC-tagged POI; (2) parallel AP–MS and BioID protein purification followed by MS analysis; and (3) protein interaction data analysis, data filtration and visualization with our localization visualization platform. The entire procedure can be completed within 25 d.
This protocol describes the MAC-tag approach, which combines affinity purification and biotinylation identification proximity labeling in a single tag. Binding proteins are identified by liquid chromatography–mass spectrometry, followed by visualization of protein localization using an online platform.
Journal Article
5-HT sub(1A) receptor pharmacophores to screen for off-target activity of alpha sub(1)-adrenoceptor antagonists
The alpha sub(1)-adrenoceptors ( alpha sub(1)-ARs), in particular the alpha sub(1A)-AR subtype, are current therapeutic targets of choice for the treatment of urogenital conditions, such as benign prostatic hyperplasia (BPH). Due to the similarity between the transmembrane domains of the alpha sub(1)-AR subtypes, and the serotonin receptor subtype 1A (5-HT sub(1A)-R), currently used alpha sub(1)-AR subtype-selective drugs to treat BPH display considerable off-target affinity for the 5-HT sub(1A)-R, leading to side effects. We describe the construction and validation of pharmacophores for 5-HT sub(1A)-R agonists and antagonists. Through the structural diversity of the training sets used in their development, these pharmacophores define the properties of a compound needed to bind to 5-HT sub(1A) receptors. Using these and previously published pharmacophores in virtual screening and profiling, we have identified unique chemical compounds (hits) that fit the requirements to bind to our target, the alpha sub(1A)-AR, selectively over the off-target, the 5-HT sub(1A)-R. Selected hits have been obtained and their affinities for alpha sub(1A)-AR, alpha sub(1B)-AR and 5-HT sub(1A)-R determined in radioligand binding assays, using membrane preparations which contain human receptors expressed individually. Three of the tested hits demonstrate statistically significant selectivity for alpha sub(1A)-AR over 5-HT sub(1A)-R. All seven tested hits bind to alpha sub(1A)-AR, with two compounds displaying K sub(i) values below 1 mu M, and a further two K sub(i) values of around 10 mu M. The insights and knowledge gained through the development of the new 5-HT sub(1A)-R pharmacophores will greatly aid in the design and synthesis of derivatives of our lead compound, and allow the generation of more efficacious and selective ligands.
Journal Article
Photo-affinity labeling (PAL) in chemical proteomics: a handy tool to investigate protein-protein interactions (PPIs)
Protein-protein interactions (PPIs) trigger a wide range of biological signaling pathways that are crucial for biomedical research and drug discovery. Various techniques have been used to study specific proteins, including affinity chromatography, activity-based probes, affinity-based probes and photo-affinity labeling (PAL). PAL has become one of the most powerful strategies to study PPIs. Traditional photocrosslinkers are used in PAL, including benzophenone, aryl azide, and diazirine. Upon photoirradiation, these photocrosslinkers (Pls) generate highly reactive species that react with adjacent molecules, resulting in a direct covalent modification. This review introduces recent examples of chemical proteomics study using PAL for PPIs.
Journal Article
Binding studies of promethazine and its metabolites with human serum albumin by high-performance affinity chromatography and molecular docking in the presence of codeine
“Purple Drank”, a soft drink containing promethazine (PMZ) and codeine (COD), has gained global popularity for its hallucinogenic effects. Consuming large amounts of this combination can lead to potentially fatal events. The binding of these drugs to plasma proteins can exacerbate the issue by increasing the risk of drug interactions, side effects, and/or toxicity. Herein, the binding affinity to human serum albumin (HSA) of PMZ and its primary metabolites [N-desmethyl promethazine (DMPMZ) and promethazine sulphoxide (PMZSO)], along with COD, was investigated by high-performance affinity chromatography (HPAC) though zonal approach. PMZ and its metabolites exhibited a notable binding affinity for HSA (%b values higher than 80%), while COD exhibited a %b value of 65%. To discern the specific sites of HSA to which these compounds were bound, displacement experiments were performed using warfarin and (S)-ibuprofen as probes for sites I and II, respectively, which revealed that all analytes were bound to both sites. Molecular docking studies corroborated the experimental results, reinforcing the insights gained from the empirical data. The in silico data also suggested that competition between PMZ and its metabolites with COD can occur in both sites of HSA, but mainly in site II. As the target compounds are chiral, the enantioselectivity for HSA binding was also explored, showing that the binding for these compounds was not enantioselective.
Journal Article
Heparin: role in protein purification and substitution with animal-component free material
Heparin is a highly sulfated polysaccharide which belongs to the family of glycosaminoglycans. It is involved in various important biological activities. The major biological purpose is the inhibition of the coagulation cascade to maintain the blood flow in the vasculature. These properties are employed in several therapeutic drugs. Heparin’s activities are associated with its interaction to various proteins. To date, the structural heparin-protein interactions are not completely understood. This review gives a general overview of specific patterns and functional groups which are involved in the heparin-protein binding. An understanding of the heparin-protein interactions at the molecular level is not only advantageous in the therapeutic application but also in biotechnological application of heparin for downstreaming. This review focuses on the heparin affinity chromatography. Diverse recombinant proteins can be successfully purified by this method. While effective, it is disadvantageous that heparin is an animal-derived material. Animal-based components carry the risk of contamination. Therefore, they are liable to strict quality controls and the validation of effective good manufacturing practice (GMP) implementation. Hence, adequate alternatives to animal-derived components are needed. This review examines strategies to avoid these disadvantages. Thereby, alternatives for the provision of heparin such as chemical synthesized heparin, chemoenzymatic heparin, and bioengineered heparin are discussed. Moreover, the usage of other chromatographic systems mimetic the heparin effect is reviewed.
Journal Article
Enhanced sulfate pseudo-affinity chromatography using monolith-like particle architecture for purifying SARS-CoV-2
Traditional virus chromatographic purification face limitations owing to the small pore sizes of conventional resins, which restrict efficient virus binding. The newly developed MLP1000 DexS, a cellulose monolith-like particle (MLP) with large continuous pores (radius of 1.5 μm) and a sulfate pseudo-affinity ligand, facilitates virus access to intraparticle surfaces and significantly enhances binding capacity. In this study, we investigated the effectiveness of MLP1000 DexS for purifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from Vero cells. Using a 0.29 mL column volume, we evaluated this resin through bind-elute mode chromatography under two load volume conditions (4.5 mL and 21 mL). MLP1000 DexS exhibited superior performance under high-loading conditions, achieving a high elution recovery of 59 % for the virus compared with that of 11–17 % for the commercial resins Cellufine Sulfate and Capto DeVirS. Additionally, the dsDNA removal capacity of MLP1000 DexS was 3.0–5.3-fold higher than that of the other resins. These findings suggest that MLP1000 DexS is an effective purification material for the downstream processing of live-attenuated and inactivated coronavirus vaccine production.
Journal Article
Synthesis and Structure-Affinity Relationships of Receptor Ligands with 1,3-Dioxane Structure
Background/Objectives: Ligands blocking σ1 receptors or NMDA receptors show promising pharmacological properties, such as analgesia or neuroprotection. It had been shown that depending on the stereochemistry and substitution pattern, 1,3-dioxnaes can selectively interact with either σ1 receptors or the phencyclidine binding site of NMDA receptors. Herein, systematic modifications of homologous aminobutyl substituted 1,3-dioxanes were conducted in order to identify ligands selectively addressing σ receptors or NMDA receptors. Methods: The first step of the synthesis, i.e., the acetalization of benzaldehyde (7a) or propiophenone (7b) with pentane-1,3,5-triol (6), determined the relative configuration of the envisaged 1,3-dioxanes bearing 4-aminobutyl substituents in 4-position. Multi-step homologation of ethanols 8 provided various primary, secondary and tertiary amines 14, 16–19, and 24–27. The affinity towards σ1 and σ2 receptors as well as the PCP and ifenprodil binding sites of the NMDA receptor was systematically evaluated in radioligand receptor binding studies. Results: Only the primary amines 14b and 24b derived from propiophenone interacted moderately with the PCP binding site of the NMDA receptor. Within this class of compounds, the N-benzylamines 17 and 18 showed the highest σ1 affinity with high selectivity over the PCP binding site and at least preference over the σ2 receptor. The benzylamine 17a (Ki(σ1) = 31 nM, LLE = 6.19) and the pyrrolidine 19a (Ki(σ1) = 154 nM, LLE = 6.72) represent the most promising σ1 ligands of this compound series, when taking the lipophilicity and receptor selectivity into account. Conclusions: Both compounds showed medium metabolic stability in vitro rendering them promising candidates for further studies.
Journal Article