Equilibrium Binding Experiments Research Articles

The Amount of Cross-Linker Influences Affinity and Selectivity of NanoMIPs Prepared by Solid-Phase Polymerization Synthesis.

The cross-linker methylene-bis-acrylamide is usually present in nanoMIPs obtained by solid-phase polymerization synthesis at 2 mol% concentration, with very few exceptions. Here, we studied the influence of variable amounts of methylene-bis-acrylamide in the range between 0 (no cross-linker) and 50 mol% concentration on the binding properties of rabbit IgG nanoMIPs. The binding parameters were determined by equilibrium binding experiments and the results show that the degree of cross-linking defines three distinct types of nanoMIPs: (i) those with a low degree of cross-linking, including nanoMIPs without cross-linker (0-05 mol%), showing a low binding affinity, high density of binding sites, and low selectivity; (ii) nanoMIPs with a medium degree of cross-linking (1-18 mol%), showing higher binding affinity, low density of binding sites, and high selectivity; (iii) nanoMIPs with a high degree of cross-linking (32-50 mol%), characterized by non-specific nanopolymer-ligand interactions, with low binding affinity, high density of binding sites, and no selectivity. In conclusion, the results are particularly relevant in the synthesis of high-affinity, high-selectivity nanoMIPs as they demonstrate that a significant gain in affinity and selectivity could be achieved with pre-polymerization mixtures containing quantities of cross-linker up to 10-20 mol%, well higher than those normally used in this technique.

One-pot synthesis of ion-imprinted three-dimensional porous material based on graphene oxide for the selective adsorption of copper(II)

Molecularly imprinted polymers (MIPs) are synthetic polymers with predetermined selectivity for a given analyte. One major problem associated with MIPs is the inaccessibility of a large fraction of the recognition sites that remain buried within the polymeric matrix. To address this problem, the high selectivity imparted by the imprinting technique and the porosity of three-dimensional (3D) graphene oxide (GO)-based porous materials were utilized in this work to prepare a 3D GO-based Cu(II)-ion-imprinted material (hereafter denoted as IIM) via one-pot reactions of GO, chitosan (CS), and glutaraldehyde in the presence of Cu(II). Results of equilibrium binding experiments show that IIM has a high template-ion binding capacity (1.75 mmol g−1) and good imprinting factor (2.19). Further, results of selectivity tests indicate that IIM has a high Cu(II)-recognition ability. IIM also has a fast binding rate and satisfactory reusability. In addition, the Langmuir isotherm model was well fitted with the experimental data, indicating the monolayer adsorption of Cu(II) ions. The present work provided a convenient approach to prepare 3D GO-based imprinted materials that are promising for enrichment or recycling of target compounds from wastewater.

Effect of Salts on the Conformational Dynamics of the Cytochrome P450 OleP.

Cytochrome P450 OleP catalytic activity is strongly influenced by its structural dynamic conformational behavior. Here, we combine equilibrium-binding experiments with all-atom molecular dynamics simulations to clarify how different environments affect OleP conformational equilibrium between the open and the closed-catalytic competent-forms. Our data clearly show that at high-ionic strength conditions, the closed form is favored, and, very interestingly, different mechanisms, depending on the chemistry of the cations, can be used to rationalize such an effect.

Curvature dependence of BAR protein membrane association and dissociation kinetics

BAR (Bin/Amphiphysin/Rvs) domain containing proteins function as lipid bilayer benders and curvature sensors, and they contribute to membrane shaping involved in cell signaling and metabolism. The mechanism for their membrane shape sensing has been investigated by both equilibrium binding and kinetic studies. In prior research, stopped-flow spectroscopy has been used to deduce a positive dependence on membrane curvature for the binding rate constant, kon, of a BAR protein called endophilin. However, the impact of bulk diffusion of endophilin, on the kinetic binding parameters has not been thoroughly considered. Employing similar methods, and using lipid vesicles of multiple sizes, we obtained a linear dependence of kon on vesicle curvature. However, we found that the observed relation can be explained without considering the local curvature sensing ability of endophilin in the membrane association process. In contrast, the diffusion-independent unbinding rate constant (koff) obtained from stopped-flow measurements shows a negative dependence on membrane curvature, which is controlled/mediated by endophilin-membrane interactions. This latter dependency, in addition to protein–protein interactions on the membrane, explains the selective binding of BAR proteins to highly curved membranes in equilibrium binding experiments.

Auxiliary ATP binding sites support DNA unwinding by RecBCD

The RecBCD helicase initiates double-stranded break repair in bacteria by processively unwinding DNA with a rate approaching ∼1,600 bp·s−1, but the mechanism enabling such a fast rate is unknown. Employing a wide range of methodologies — including equilibrium and time-resolved binding experiments, ensemble and single-molecule unwinding assays, and crosslinking followed by mass spectrometry — we reveal the existence of auxiliary binding sites in the RecC subunit, where ATP binds with lower affinity and distinct chemical interactions as compared to the known catalytic sites. The essentiality and functionality of these sites are demonstrated by their impact on the survival of E.coli after exposure to damage-inducing radiation. We propose a model by which RecBCD achieves its optimized unwinding rate, even when ATP is scarce, by using the auxiliary binding sites to increase the flux of ATP to its catalytic sites.

The fabrication of molecularly imprinted polymer microspheres via Pickering emulsion polymerization stabilized with novel ferric hydroxide colloid

In this paper, the ferric hydroxide colloid was prepared firstly, which was found suitable for the stabilizer of Pickering emulsion because of proper hydrophilicity. Then, molecularly imprinted polymers (MIPs) microspheres were synthesized by means of facile Pickering emulsion polymerization using ferric hydroxide colloid as emulsifier. In the synthesis process, hydrophilic methacrylic acid was used for the functional monomer, and ethylene glycol dimethacrylate served as the crosslinking agent. Bisphenol A (BPA), a typical endocrine-disrupting chemical, was used for the template molecules. The whole synthesis was based on the hydrogen bonds interaction between the functional monomer and template molecule. The ferric hydroxide colloid could be removed from the surface of microspheres by gentle washing. The morphology of obtained MIPs microspheres were assessed through the optical microscope and field emission scanning electron microscope, respectively. The results suggested that the MIPs appeared the regular spherical form with relatively wide size distribution, whose diameter varied from 10 to 60 μm. The adsorptive property was studied detailedly through a series of rebinding experiments. The research showed that MIPs have fast binding kinetics, whose rebinding equilibrium in tested concentration could be achieved within 60 min, and the rebinding capacity in 0.3 mmol L−1 of BPA initial solution is 8.19 μmol g−1. Additionally, the binding capacities of MIPs were larger than that of non-imprinted polymers in the batch equilibrium binding experiments. The binding selectivity was investigated with 4, 4′-biphenol and hydroquinone, which played the role of the structurally similar compounds of template molecules. The result showed that the MIPs were highly selective for BPA, and the imprinting factor was calculated to be 1.63. Reusability experiment indicates that our prepared MIPs have good mechanical stability and could be reused at least 5 times. The MIPs microspheres would be valuable in the field of environmental protection.

LANCL1 binds abscisic acid and stimulates glucose transport and mitochondrial respiration in muscle cells via the AMPK/PGC-1α/Sirt1 pathway

ObjectiveAbscisic acid (ABA) is a plant hormone also present and active in animals. In mammals, ABA regulates blood glucose levels by stimulating insulin-independent glucose uptake and metabolism in adipocytes and myocytes through its receptor LANCL2. The objective of this study was to investigate whether another member of the LANCL protein family, LANCL1, also behaves as an ABA receptor and, if so, which functional effects are mediated by LANCL1. MethodsABA binding to human recombinant LANCL1 was explored by equilibrium-binding experiments with [3H]ABA, circular dichroism, and surface plasmon resonance. Rat L6 myoblasts overexpressing either LANCL1 or LANCL2, or silenced for the expression of both proteins, were used to investigate the basal and ABA-stimulated transport of a fluorescent glucose analog (NBDG) and the signaling pathway downstream of the LANCL proteins using Western blot and qPCR analysis. Finally, glucose tolerance and sensitivity to ABA were compared in LANCL2−/− and wild-type (WT) siblings. ResultsHuman recombinant LANCL1 binds ABA with a Kd between 1 and 10 μM, depending on the assay (i.e., in a concentration range that lies between the low and high-affinity ABA binding sites of LANCL2). In L6 myoblasts, LANCL1 and LANCL2 similarly, i) stimulate both basal and ABA-triggered NBDG uptake (4-fold), ii) activate the transcription and protein expression of the glucose transporters GLUT4 and GLUT1 (4-6-fold) and the signaling proteins AMPK/PGC-1α/Sirt1 (2-fold), iii) stimulate mitochondrial respiration (5-fold) and the expression of the skeletal muscle (SM) uncoupling proteins sarcolipin (3-fold) and UCP3 (12-fold). LANCL2−/− mice have a reduced glucose tolerance compared to WT. They spontaneously overexpress LANCL1 in the SM and respond to chronic ABA treatment (1 μg/kg body weight/day) with an improved glycemia response to glucose load and an increased SM transcription of GLUT4 and GLUT1 (20-fold) of the AMPK/PGC-1α/Sirt1 pathway and sarcolipin, UCP3, and NAMPT (4- to 6-fold). ConclusionsLANCL1 behaves as an ABA receptor with a somewhat lower affinity for ABA than LANCL2 but with overlapping effector functions: stimulating glucose uptake and the expression of muscle glucose transporters and mitochondrial uncoupling and respiration via the AMPK/PGC-1α/Sirt1 pathway. Receptor redundancy may have been advantageous in animal evolution, given the role of the ABA/LANCL system in the insulin-independent stimulation of cell glucose uptake and energy metabolism.

Targeting the Interaction between the SH3 Domain of Grb2 and Gab2.

Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction.

Sulfonated calix[4]arene functionalized SiO2@TiO2 for recognition of lysine methylation

Lysine methylations are common protein post-translational modifications (PTMs), that play significant roles in regulating gene activities. Studies of their functions and connections with diseases have important values. However, due to the small variations from their native structures and very low component proportions, it is very difficult to extract methylated peptides from biological mixtures. In this research, a new material that utilizes sulfonated calix[4]arene (SC4A) as the recognition unit and silica coated with TiO2 as carrier, denoted as SiO2@TiO2@SC4A, was synthesized. The equilibrium binding experiments demonstrated that SiO2@TiO2@SC4A can identify lysine and arginine methylation and peptides with these methylated residues. The maximum isotherm binding capacities are 70.0, 55.9, 31.4 and 24.8 μmol g−1 for Lys(Me3), Lys(Me)2, Lys(Me) and Lys, respectively. It demonstrated that the higher the degree of methylation, the stronger the interactions.In addition, the analyses of high performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) demonstrated that peptides with methylated lysine or arginine can be selectively extracted from spiked histone trypsin digestion. The recoveries for the spiked GGAK(Me)R, GGAKR(Me)2 and GGAK(Me)3R are 83%, 78%, and 84% respectively. The experiments from the nuclear extracts of HeLa cells also illustrated that SiO2@TiO2@SC4A holds a potential in the enrichment and identification of lysine methylations.

Carbachol dimers with primary carbamate groups as homobivalent modulators of muscarinic receptors

Although agonists and antagonists of muscarinic receptors have been known for long time, there is renewed interest in compounds (such as allosteric or bitopic ligands, or biased agonists) able to differently and selectively modulate these receptors. As a continuation of our previous research, we designed a new series of dimers of the well-known cholinergic agonist carbachol. The new compounds were tested on the five cloned human muscarinic receptors (hM1–5) expressed in CHO cells by means of equilibrium binding experiments, showing a dependence of the binding affinity on the length and position of the linker connecting the two monomers. Kinetic binding studies revealed that some of the tested compounds were able to slow the rate of NMS dissociation, suggesting allosteric behavior, also supported by docking simulations. Assessment of ERK1/2 phosphorylation on hM1, hM2 and hM3 activation showed that the new compounds are endowed with muscarinic antagonist properties. At hM2 receptors, some compounds were able to stimulate GTPγS binding but not cAMP accumulation, suggesting a biased behavior.Classification, Molecular and cellular pharmacology.

Extraction of tetracycline in food samples using biochar microspheres prepared by a Pickering emulsion method

We report for the first time the use of biochar as a stabilizer for oil-in-water (o/w) Pickering emulsion. The emulsion is subsequently used to prepare tetracycline-imprinted biochar composite microspheres (MIPMs) with tailored sizes and good uniformity. The adsorption properties of tetracycline to the MIPMs were investigated using different adsorption experiments including adsorption kinetic experiment, equilibrium binding experiment, selectivity evaluation and competitive adsorption tests. The MIPMs were used as adsorbent for solid phase extraction (SPE) for the extraction of tetracycline present in drinking water, fish, and chicken samples. Under optimal conditions, the results showed good recovery yield ranging from ~73% to ~95% with a relative standard deviation (RSD) ranging from ~0.3% to ~8.4%, respectively.

A Mild Method for Preparation of Highly Selective Magnetic Biochar Microspheres.

We report the use of biochar and Fe3O4 nanoparticles as co-stabilizers for oil-in-water (o/w) Pickering emulsion. The emulsion is subsequently used to prepare magnetic tetracycline-imprinted biochar composite microspheres (MMIPMs) with good uniformity and high selectivity. The MMIPMs were characterized by scanning electron microscopy (SEM), Brunner-Emmet-Teller (BET) measurements, Fourier transform infrared spectrometry (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM) and thermogravimetry analysis (TGA). The adsorption properties of tetracycline to the MMIPMs were investigated using different adsorption experiments including adsorption kinetic experiment, equilibrium binding experiment, selectivity evaluation and competitive adsorption tests. The theoretical maximum adsorption capacity of the MMIPMs (15.45 mg g−1) was greater than that of the raw biochar (2.10 mg g−1) and non-imprinted biochar composite microspheres (3.39 mg g−1) for tetracycline. Further, the MMIPMs were used as adsorbent for magnetic solid phase extraction (SPE) for the extraction of tetracycline present in drinking water, milk, fish and chicken samples. Under optimal conditions, the results showed good recovery yield ranging from 88.41% to 106.29% with a relative standard deviation (RSD) ranging from 0.35% to 6.83%, respectively.

Synthesis of molecularly imprinted polymer adsorbents for solid-phase extraction of strobilurin fungicides from agricultural products.

Molecularly imprinted polymers for strobilurin fungicides were prepared by precipitation polymerization employing azoxystrobin as template molecular together with methacrylic acid monomer and trimethylolpropane triacrylate cross-linker. Morphological characterization showed molecularly imprinted polymers were uniform spherical particles with about 0.2 μm in diameter, while the morphologies of nonimprinted polymers were irregular bulk. The equilibrium binding and selective experiments proved that molecularly imprinted polymers possessed a higher affinity toward four fungicides compared to nonimprinted polymers and heterogeneous binding sites were found in the molecularly imprinted polymers. Molecularly imprinted solid-phase extraction conditions, including sample loading solvents, selective washing, and elution solvents, were carefully optimized. The developed method showed good recoveries (70.0-114.0%) with relative standard deviations in range of 1.0-9.8% (n = 3) for samples (cucumber and peach) spiked at three different levels (10, 50, and 100 μg/ kg). The detection limit (signal/noise=3) ranged from 0.01 to 0.08 μg/kg. The results demonstrated good potential use of this convenient and highly efficient method for determining trace strobilurin fungicides in agricultural products.

Characterzing the Auxiliary Nucleotide Binding Sites in RecBCD DNA Helicase, Towards Deciphering their Functional Role

RecBCD is a DNA helicase-nuclease, powering the initiation of ds break repair. It is a highly processive fast helicase, with unwinding rates approaching 1,600 bp/s. By employing a combination of equilibrium and time-resolved unwinding and binding experiments(both ensemble and single-molecule) to unravel RecBCD's molecular mechanism, we had demonstrated the existence of auxiliary binding sites in RecBCD, specifically in the RecC subunit, where ATP binds with lower affinity and with distinct chemical interactions as compared to the known catalytic sites. According to our model, RecBCD achieves its fast unwinding rate by utilizing the auxiliary binding sites to increase the flux of ATP to its catalytic sites. The number of nucleotide-binding sites determined by equilibrium dialysis has been estimated to be at least four. While RecB and RecD, each have one known structurally and chemically well-defined nucleotide-binding site, the additional two (or more) auxiliary nucleotide-binding sites have not been located. Our current study strongly supports that they are likely to be located in RecC. In this work, we have attempted to abolish the nucleotide-binding sites in RecC by using a multi-step approach. A list of possible nucleotide-binding sites was obtained by using a combination of UV-crosslinking, mass spectrometry and molecular docking studies. Based on this, several mutations in RecC were carefully designed and successfully cloned and purified to produce RecBCmutD. RecBCmutD has an activated DNA ATPase activity, with a higher KM,ATP. Furthermore, equilibrium nucleotide binding assay suggests that the RecBCmutD does not display the same biphasic binding isotherm as the WT RecBCD. Based on this, we propose that the auxiliary binding sites play an integrated role in the reaction mechanism of RecBCD.

Selective removal of congo red from wastewater using molecularly imprinted polymer

ABSTRACT Molecular imprinted polymer microspheres (MIP) were prepared by a one-step swelling polymerization method using polystyrene microspheres as seeds, congo red (CR) as template molecules, methacrylic acid as the functional monomer, methyl acrylic acid ethylene glycol ester as cross-linker, acetonitrile as the solvent and azobisisobutyronitrile as initiator. The morphology and structure of MIP were characterized by SEM and FT-IR. The selective recognition ability of MIP was investigated using the equilibrium binding experiments. Results showed that the adsorption process could be depicted by the pseudo-second-order model. Relative to competitive analogs of direct yellow GR and methyl orange, the selectivity coefficients of CR-MIP for congo red are 2.163 and 4.908, respectively. CR-MIP could be employed as an effective material for removing congo red from wastewater.

Synthesis of molecularly imprinted polymers using a functionalized initiator for chiral-selective recognition of propranolol.

We present a new concept of synthesis for preparation of molecularly imprinted polymers using a functionalized initiator to replace the traditional functional monomer. Using propranolol as a model template, a carboxyl‐functionalized radical initiator was demonstrated to lead to high‐selectivity polymer particles prepared in a standard precipitation polymerization system. When a single enantiomer of propranolol was used as template, the imprinted polymer particles exhibited clear chiral selectivity in an equilibrium binding experiment. Unlike the previous molecular imprinting systems where the active free radicals can be distant from the template‐functional monomer complex, the method reported in this work makes sure that the actual radical polymerization takes place in the vicinity of the template‐associated functional groups. The success of using functional initiator to synthesize molecularly imprinted polymers brings in new possibilities to improve the functional performance of molecularly imprinted synthetic receptors.

Exploring the molecular basis for substrate specificity in homologous macrolide biosynthetic cytochromes P450

Cytochromes P450 (P450s) are nature's catalysts of choice for performing demanding and physiologically vital oxidation reactions. Biochemical characterization of these enzymes over the past decades has provided detailed mechanistic insight and highlighted the diversity of substrates P450s accommodate and the spectrum of oxidative transformations they catalyze. Previously, we discovered that the bacterial P450 MycCI from the mycinamicin biosynthetic pathway in Micromonospora griseorubida possesses an unusually broad substrate scope, whereas the homologous P450 from tylosin-producing Streptomyces fradiae (TylHI) exhibits a high degree of specificity for its native substrate. Here, using biochemical, structural, and computational approaches, we aimed to understand the molecular basis for the disparate reactivity profiles of these two P450s. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI strictly prefers 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. To help rationalize these results, we solved the X-ray crystal structure of TylHI in complex with its native substrate at 1.99-Å resolution and assayed several site-directed mutants. We also conducted molecular dynamics simulations of TylHI and MycCI and biochemically characterized a third P450 homolog from the chalcomycin biosynthetic pathway in Streptomyces bikiniensis These studies provided a basis for constructing P450 chimeras to gain further insight into the features dictating the differences in reaction profile among these structurally and functionally related enzymes, ultimately unveiling the central roles of key loop regions in influencing substrate binding and turnover. Our work highlights the complex nature of P450/substrate interactions and raises interesting questions regarding the evolution of functional diversity among biosynthetic enzymes.

Allosteric feedback inhibition of pyridoxine 5′-phosphate oxidase from Escherichia coli

In Escherichia coli, the synthesis of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, takes place through the so-called deoxyxylulose 5-phosphate-dependent pathway, whose last step is pyridoxine 5'-phosphate (PNP) oxidation to PLP, catalyzed by the FMN-dependent enzyme PNP oxidase (PNPOx). This enzyme plays a pivotal role in controlling intracellular homeostasis and bioavailability of PLP. PNPOx has been proposed to undergo product inhibition resulting from PLP binding at the active site. PLP has also been reported to bind tightly at a secondary site, apparently without causing PNPOx inhibition. The possible location of this secondary site has been indicated by crystallographic studies as two symmetric surface pockets present on the PNPOx homodimer, but this site has never been verified by other experimental means. Here, we demonstrate, through kinetic measurements, that PLP inhibition is actually of a mixed-type nature and results from binding of this vitamer at an allosteric site. This interpretation was confirmed by the characterization of a mutated PNPOx form, in which substrate binding at the active site is heavily hampered but PLP binding is preserved. Structural and functional connections between the active site and the allosteric site were indicated by equilibrium binding experiments, which revealed different PLP-binding stoichiometries with WT and mutant PNPOx forms. These observations open up new horizons on the mechanisms that regulate E. coli PNPOx, which may have commonalities with the mechanisms regulating human PNPOx, whose crucial role in vitamin B6 metabolism and epilepsy is well-known.

Conformational Dynamics and Cooperativity Drive the Specificity of a Protein-Ligand Interaction

Molecular recognition is critical for the fidelity of signal transduction in biology. Conversely, the disruption of protein-protein interactions can lead to disease. Thus, comprehension of the molecular determinants of specificity is essential for understanding normal biological signaling processes and for the development of precise therapeutics. Although high-resolution structures have provided atomic details of molecular interactions, much less is known about the influence of cooperativity and conformational dynamics. Here, we used the Tiam2 PSD-95/Dlg/ZO-1 (PDZ) domain and a quadruple mutant (QM), engineered by swapping the identity of four residues important for specificity in the Tiam1 PDZ into the Tiam2 PDZ domain, as a model system to investigate the role of cooperativity and dynamics in PDZ ligand specificity. Surprisingly, equilibrium binding experiments found that the ligand specificity of the Tiam2 QM was switched to that of the Tiam1 PDZ. NMR-based studies indicated that Tiam2 QM PDZ, but not other mutants, had extensive microsecond to millisecond motions distributed throughout the entire domain suggesting structural cooperativity between the mutated residues. Thermodynamic analyses revealed energetic cooperativity between residues in distinct specificity subpockets that was dependent upon the identity of the ligand, indicating a context-dependent binding mechanism. Finally, isothermal titration calorimetry experiments showed distinct entropic signatures along the mutational trajectory from the Tiam2 wild-type to the QM PDZ domain. Collectively, our studies provide unique insights into how structure, conformational dynamics, and thermodynamics combine to modulate ligand-binding specificity and have implications for the evolution, regulation, and design of protein-ligand interactions.

Trace analysis of estrogens in milk samples by molecularly imprinted solid phase extraction with genistein as a dummy template molecule and high-performance liquid chromatography–tandem mass spectrometry

Dummy molecularly imprinted polymer microspheres (DMIPMS) towards estrogens were synthesized by Pickering emulsion polymerization employing genistein (GEN) as a dummy template molecule. The FTIR analysis indicated the successful preparation of the imprinted polymers, and the characterization results of scanning electron microscopy and nitrogen adsorption desorption measurement indicated that the obtained DMIPMS are in possess of regular spherical shapes, porous structures and narrow diameter distribution, a BET surface area of 402.74 m2 g−1, a total pore volume of 0.568 cm3 g−1 and a pore diameter of 3.62 nm. The binding capacity and selectivity of DMIPMS were investigated in equilibrium binding experiments and chromatographic evaluation experiments through scatchard analysis and molecular imprinting factor (IF) analysis, respectively. The MIPs showed high binding capacity and excellent selectivity towards seven selected natural and synthetic estrogens, which are Estrone (E1), 17β-estradiol (βE2), estriol (E3), ethinylestradiol (EE2), dienestrol(DS), diethylstilbestrol (DES), and hexestrol (HEX). A method for selective determination of seven estrogens in milk samples via dummy molecularly imprinted solid phase extraction coupled with HPLC-MS/MS was developed, which showed good linearity from 2 to 500 µg L−1 with a correlation coefficient (R2) of more than 0.999. The detection limits were within the range of 0.10–0.35 µg L−1 and the recoveries of the seven estrogens at three spiking levels (10,100,250 µg L−1) ranged from 88.9% to 102.3% with relative standard deviation (RSD, n = 5) for intra-day and inter-day assays varied from 0.8% to 4.5%. The developed method is thus proven to be efficient and reliable for regular monitoring of trace estrogens in complex matrices such as milk samples.