Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS.     

Identification of a lacosamide binding protein using an affinity bait and chemical reporter strategy: 14-3-3 ζ

We have advanced a useful strategy to elucidate binding partners of ligands (drugs) with modest binding affinity. Key to this strategy is attaching to the ligand an affinity bait (AB) and a chemical reporter (CR) group, where the AB irreversibly attaches the ligand to the receptor upon binding and the CR group is employed for receptor detection and isolation. We have tested this AB&CR strategy using lacosamide ((R)-1), a low-molecular-weight antiepileptic drug. We demonstrate that using a (R)-lacosamide AB&CR agent ((R)-2) 14-3-3 ζ in rodent brain soluble lysates is preferentially adducted, adduction is stereospecific with respect to the AB&CR agent, and adduction depends upon the presence of endogenous levels of the small molecule metabolite xanthine. Substitution of lacosamide AB agent ((R)-5) for (R)-2 led to the identification of the 14-3-3 ζ adduction site (K120) by mass spectrometry. Competition experiments using increasing amounts of (R)-1 in the presence of (R)-2 demonstrated that (R)-1 binds at or near the (R)-2 modification site on 14-3-3 ζ. Structure-activity studies of xanthine derivatives provided information concerning the likely binding interaction between this metabolite and recombinant 14-3-3 ζ. Documentation of the 14-3-3 ζ-xanthine interaction was obtained with isothermal calorimetry using xanthine and the xanthine analogue 1,7-dimethylxanthine.     

Ultrasensitive competitive electrochemiluminescence immunoassay for the β-adrenergic agonist phenylethanolamine A using quantum dots and enzymatic amplification

Microchimica Acta - We have designed an ultrasensitive electrochemiluminescence (ECL) immunoassay for the determination of the β-adrenergic agonist phenylethanolamine A (PA). It is based on...     

Interactions between β-cyclodextrin and an amino acid-based anionic gemini surfactant derived from cysteine

The interaction between β-cyclodextrin (β-CD) and an amino acid-based anionic gemini surfactant derived from cysteine (C₈Cys)₂ was studied by three independent techniques: electrical conductivity, UV–Vis spectral displacement technique using phenolphthalein as probe, and ¹H NMR spectroscopy. The data obtained indicated the formation of a 1:1 inclusion complex between β-CD and the gemini surfactant studied and allowed for the determination of the binding constant, K₁, by considering this stoichiometry. Electrical conductivity, spectral displacement technique, and NMR chemical shift measurements, obtained for aqueous β-CD–surfactant systems, yielded consistent K₁ values in the order of 10² dm³ mol^?1, typical of a weakly bound β-CD–surfactant complex. The influence of the presence of the inclusion complex on the micellization process of the gemini surfactant has also been studied and the apparent critical micelle concentration (cmc¹) has been obtained. Increasing β-CD concentration was found to shift the cmc¹ to higher values, as complexed surfactant monomers are not available to form micelles and aggregation takes place only when all β-CD cavities are occupied.     

Electrochemical signal modulation in homogeneous solutions using the formation of an inclusion complex between ferrocene and β-cyclodextrin on a DNA scaffold

Two DNA conjugates modified with ferrocene and β-cyclodextrin were prepared as a pair of probes that work cooperatively for DNA sensing, in which the electrochemical signal of ferrocene on one probe was significantly "quenched" by the formation of an inclusion complex with β-cyclodextrin of the other probe on the DNA templates.     

Thermodynamic studies of inclusion complex formation between alkylpyridinium chlorides and β-cyclodextrin using conductometric method

Thermodynamics on inclusion complexation of β-cyclodextrin (β-CD) with n-alkylpyridinium chlorides (C _n PC, n = 12, 14, 16) were measured by conductivity technique to evaluate the effects of chain length of C _n PC and temperature. The data obtained indicate that inclusion complexes S(CD) and S(CD)₂ had formed between surfactant and β-CD in aqueous solution. Investigation showed that the K ₁ (first equilibrium constant) for S(CD) formation is greater than K ₂ (second equilibrium constant) for S(CD)₂ formation. It has been found that C₁₂PC forms only the 1:1 complex, while C₁₄PC and C₁₆PC form 1:1 and 1:2 complexes. Thermodynamic parameters of the complexation, i.e. ΔG°, ΔH° and ΔS° have been also calculated. The large values of ΔG° indicate that complexation between surfactant and β-CD is very favorable.     

A multistage pathway for human prion protein aggregation in vitro: from multimeric seeds to β-oligomers and nonfibrillar structures

Aberrant protein aggregation causes numerous neurological diseases including Creutzfeldt-Jakob disease (CJD), but the aggregation mechanisms remain poorly understood. Here, we report AFM results on the formation pathways of β-oligomers and nonfibrillar aggregates from wild-type full-length recombinant human prion protein (WT) and an insertion mutant (10OR) with five additional octapeptide repeats linked to familial CJD. Upon partial denaturing, seeds consisting of 3-4 monomers quickly appeared. Oligomers of ~11-22 monomers then formed through direct interaction of seeds, rather than by subsequent monomer attachment. All larger aggregates formed through association of these β-oligomers. Although both WT and 10OR exhibited identical aggregation mechanisms, the latter oligomerized faster due to lower solubility and, hence, thermodynamic stability. This novel aggregation pathway has implications for prion diseases as well as others caused by protein aggregation.     

Determination of primary structure and microheterogeneity of a β-amyloid plaque-specific antibody using high-performance LC–tandem mass spectrometry

Using the bottom-up approach and liquid chromatography (LC) in combination with mass spectrometry, the primary structure and sequence microheterogeneity of a plaque-specific anti-β-amyloid (1–17) monoclonal antibody (clone 6E10) was characterized. This study describes the extent of structural information directly attainable by a high-performance LC–tandem mass spectrometric method in combination with both protein database searching and de novo sequence determination. Using trypsin and chymotrypsin for enzymatic digestion, 95% sequence coverage of the light chain and 82% sequence coverage of the heavy chain of the 6E10 antibody were obtained. The primary structure determination of a large number of peptides from the antibody variable regions was obtained through de novo interpretation of the data. In addition, N-terminal truncations of the heavy chain were identified as well as low levels of pyroglutamic acid formation. Surprisingly, pronounced sequence microheterogeneities were determined for the CDR 2 region of the light chain, indicating that changes at the protein level derived from somatic hypermutation of the Ig V_L genes in mature B-cells might contribute to unexpected structural diversity. Furthermore, the major glycoforms at the conserved heavy chain N-glycosylation site, Asn-292, were determined to be core-fucosylated, biantennary, complex-type structures containing zero to two galactose residues. Figure Primary structure and sequence microheterogeneities of a β-amyloid plaque-specific monoclonal antibody were identified by high-performance LC-tandem-mass spectrometry. Sequence heterogeneities of the light chain CDR2 reveal unexpected diversity from VL-gene hypermutations. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Synthesis and protein binding studies of a peptide fragment of clathrin assembly protein AP180 bearing an O-linked β-N-acetylglucosaminyl-6-phosphate modification

A novel post-translational modification of threonine, β-N-acetylglucosaminyl-phosphate, was recently discovered on assembly protein AP180, a protein which plays a crucial role in clathrin coated vesicle formation in synaptic vesicle endocytosis (SVE). Herein, we report studies aimed at probing the effect of this modification on binding to proteins in rat brain lysate using pull down experiments with peptide fragments of AP180.     

Solvent reaction field potential inside an uncharged globular protein: a bridge between implicit and explicit solvent models?

The solvent reaction field potential of an uncharged protein immersed in simple point charge/extended explicit solvent was computed over a series of molecular dynamics trajectories, in total 1560 ns of simulation time. A finite, positive potential of 13-24 kbTec(-1) (where T=300 K), dependent on the geometry of the solvent-accessible surface, was observed inside the biomolecule. The primary contribution to this potential arose from a layer of positive charge density 1.0 A from the solute surface, on average 0.008 ec/A3, which we found to be the product of a highly ordered first solvation shell. Significant second solvation shell effects, including additional layers of charge density and a slight decrease in the short-range solvent-solvent interaction strength, were also observed. The impact of these findings on implicit solvent models was assessed by running similar explicit solvent simulations on the fully charged protein system. When the energy due to the solvent reaction field in the uncharged system is accounted for, correlation between per-atom electrostatic energies for the explicit solvent model and a simple implicit (Poisson) calculation is 0.97, and correlation between per-atom energies for the explicit solvent model and a previously published, optimized Poisson model is 0.99.     

Release characteristic and stability of curcumin incorporated in β-chitin non-woven fibrous sheet using Tween 20 as an emulsifier

β-Chitin sheets containing curcumin—a naturally occurring substance that possesses several advantages biological properties including antioxidant, antimicrobial, and anti-inflammatory activities—were fabricated by the paper-making process using a water-based system. Using the chitin matrix consisting of small fibers could give rise to a large surface area that could improve the diffusion of solvent and reagent, so as to make the material suitable for use as support and carrier for drugs. Tween 20 was used as an emulsifier to improve water solubility of the curcumin. The change in surface morphology of the fabricated chitin sheets after curcumin loading was indicated by scanning electron microscope (SEM). A rough surface consisting of fibrous chitin could be seen both on the neat chitin sheets and the curcumin-loaded chitin sheets, while the increase in the curcumin content led the sheets to show an occurrence of spots. Investigation of the release behavior of the curcumin loaded into the chitin sheet was carried out by the total immersion method in an acetate buffer solution, pH 5.5, at 37 °C (simulating human skin). It was found that the amounts of loaded curcumin affected the release characteristics of the curcumin from the chitin sheet as a function of releasing time. In addition, the Tween 20 played an important role in the release ability of the curcumin to an exterior solution and in the stability of the curcumin present in the chitin sheet. It could be postulated that the water solubility, release ability, and stability of the curcumin incorporated into the β-chitin sheets was improved by the inclusion of curcumin into the cores of the Tween 20 micelles and the β-chitin non-woven fibrous sheet containing curcumin could be a promising candidate for would care materials.     

Efficient synthesis of 3-hydroxymethylated cis- and trans-cyclobutane β-amino acids using an intramolecular photocycloaddition strategy

Uracil bearing a tethered allyl alcohol appendage at N1 undergoes a [2+2] photocycloaddition reaction to provide a single tricyclic adduct in high yield. This compound is transformed in one step into a cis-cyclobutane β-amino acid bearing a 3-hydroxymethyl group. Through appropriate functionalization and epimerization, the trans isomer is obtained therefrom in only three further steps.     

A new actinomycin Z analogue with an additional oxygen bridge between chromophore and β-depsipentapeptide from Streptomyces sp. KIB-H714

Actinomycin Z₆ (1), a new member of the actinomycin family, along with three congeners of the Z-type (Z₁, Z₃, Z₅) actinomycins, are produced from Streptomyces sp. KIB-H714. Their structures were authenticated by comprehensive spectroscopic data interpretation. Different from all the reported Z-type actinomycins, the β-ring of the new compound actinomycin Z₆ includes an additional ring linked between the actinoyl chromophore and β-peptidolactone. In Z₃ and Z₅, the L-threonine in β-depsipeptide is replaced by the unusual 4-chlorothreonine, an amino acid rarely found in actinomycin family. All isolates were evaluated for cytotoxicity against five human tumor cell lines and for inhibitory activity against Candida albicans and Staphylococcus aureus.     

An approach towards the formation of an ester bond between the primary hydroxyl of a β-D-galactopyranoside and 2-aminoethylphosphonic acid and N-methyl substituted derivatives

An expeditious method for the preparation of the phosphonylating reagent 2-bromoethylphosphonic acid is presented. The latter compound as well as salicylchlorophosphite proved to be suitable for the synthesis of methyl 0-6-(2′-aminoethylphosphonyl)-β-D-galactopyranoside and N-mono- or dimethylated derivatives thereof.     

pH Sensitive supramolecular assembling system between a new linear water soluble β-cyclodextrin terpolymer and an amphiphilic poly(ethylene oxide)

A linear water soluble β-cyclodextrin terpolymer (P(MVE-AM)-g-βCD) was synthesized by grafting different amounts of βCD on the copolymer P(MVE-AM). The resulting terpolymer showed a pH sensitive behaviour in aqueous solution, due to the presence of carboxylic acid groups. Whatever the substitution degree in βCD, the terpolymer behaved like a polyacid at pH 7, and had neutral properties at pH 2. The mixture of P(MVE-AM)-g-βCD with an amphiphilic poly(ethylene oxide) (PEO-Ad₃) showed an associative phase separation influenced by the pH and concentration of the medium. In the dilute range and intermediate range, associative phase separations were induced reversibly by decreasing the pH from 7 to 2. The results in the semi-dilute range showed that networks were elaborated with much stronger dynamic modulus at low pH (pH 2) than at higher pH (pH 7). Coupled inclusion complex interactions (between the adamantyl groups of PEO-Ad₃ and the cyclodextrin cavities of P(MVE-AM)-g-βCD) with hydrogen bonding (between carboxylic acid groups and PEO backbone) could be a way to modulate the strength of the interactions in supramolecular assemblies. These results suggest that the mixture of P(MVE-AM)-g-βCD with PEO-Ad₃ could be a potential pH sensitive system.     

Constrained photophysics of an ESIPT probe within β-cyclodextrin nanocavity and chaotrope-induced perturbation of the binding phenomenon: implication towards hydrophobic interaction mechanism between urea and the molecular probe

Geological sequestration of pure carbon dioxide (CO(2)) in coal is one of the methods to sequester CO(2). In addition, injection of CO(2) or flue gas into coal enhances coal bed methane production (ECBM). The success of this combined process depends strongly on the wetting behavior of the coal, which is function of coal rank, ash content, heterogeneity of the coal surface, pressure, temperature and composition of the gas. The wetting behavior can be evaluated from the contact angle of a gas bubble, CO(2) or flue gas, on a coal surface. In this study, contact angles of a synthetic flue gas, i.e. a 80/20 (mol%) N(2)/CO(2) mixture, and pure CO(2) on a Warndt Luisenthal (WL) coal have been determined using a modified pendant drop cell in a pressure range from atmospheric to 16 MPa and a constant temperature of 318 K. It was found that the contact angles of flue gas on WL coal were generally smaller than those of CO(2). The contact angle of CO(2) changes from water-wet to gas-wet by increasing pressure above 8.5 MPa while the one for the flue gas changes from water-wet to intermediate-wet by increasing pressure above 10 MPa.     

Multi-target bioactive compound screening from the infructescence of Platycarya strobilacea Sieb. et Zucc. by affinity chromatography using immobilized β2-adrenoceptor and muscarinic-3 acetylcholine receptor as the stationary phase

As a main source for the recognition and identification of lead compounds, traditional Chinese medicine plays a pivotal role in preventing diseases for years. However, screening bioactive compounds from traditional Chinese medicine remains challenging because of the complexity of the systems and the occurrence of the synergic effect of the compounds. The infructescence of Platycarya strobilacea Sieb. et Zucc is prescribed for allergic rhinitis treatment with unknown bioactive compounds and unclear mechanisms. Herein, we immobilized the β₂-adrenoceptor and muscarine-3 acetylcholine receptor onto the silica gel surface to prepare the stationary phase in a covalent bond through one step. The feasibility of the columns was investigated by the chromatographic method. Ellagic acid and catechin were identified as the bioactive compounds targeting the receptors. The binding constants of ellagic acid were calculated to be (1.56 ± 0.23)×10⁷ M⁻¹ for muscarine-3 acetylcholine receptor and (2.93 ± 0.15)×10⁷ M⁻¹ for β₂-adrenoceptor by frontal analysis. While catechin can bind with muscarine-3 acetylcholine receptor with an affinity of (3.21 ± 0.05)×10⁵ M⁻¹. Hydrogen bonds and van der Waals’ force were the main driving forces for the two compounds with the receptors. The established method provides an alternative for multi-target bioactive compound screening in complex matrices.