Enhancing protein dynamics analysis with hydrophilic polyethylene glycol cross-linkers.

Cross-linkers play a critical role in capturing protein dynamics in chemical cross-linking mass spectrometry techniques. Various types of cross-linkers with different backbone features are widely used in the study of proteins. However, it is still not clear how the cross-linkers' backbone affect their own structure and their interactions with proteins. In this study, we systematically characterized and compared methylene backbone and polyethylene glycol (PEG) backbone cross-linkers in terms of capturing protein structure and dynamics. The results indicate the cross-linker with PEG backbone have a better ability to capture the inter-domain dynamics of calmodulin, adenylate kinase, maltodextrin binding protein and dual-specificity protein phosphatase. We further conducted quantum chemical calculations and all-atom molecular dynamics simulations to analyze thermodynamic and kinetic properties of PEG backbone and methylene backbone cross-linkers. Solution nuclear magnetic resonance was employed to validate the interaction interface between proteins and cross-linkers. Our findings suggest that the polarity distribution of PEG backbone enhances the accessibility of the cross-linker to the protein surface, facilitating the capture of sites located in dynamic regions. By comprehensively benchmarking with disuccinimidyl suberate (DSS)/bis-sulfosuccinimidyl-suberate(BS3), bis-succinimidyl-(PEG)2 revealed superior advantages in protein dynamic conformation analysis in vitro and in vivo, enabling the capture of a greater number of cross-linking sites and better modeling of protein dynamics. Furthermore, our study provides valuable guidance for the development and application of PEG backbone cross-linkers.

Crosslinker Nanocarriers Delivery to Chloroplasts for In Vivo Mapping of Photosynthetic Membrane Protein Complexes in Living Chlamydomonas reinhardtii Cells.

Photosynthesis, as the core of solar energy biotransformation, is driven by photosynthetic membrane protein complexes in plants and algae. Current methods for intracellular photosynthetic membrane protein complex analysis mostly require the separation of specific chloroplasts or the change of the intracellular environment, which causes the missing of real-time and on-site information. Thus, we explored a method for in vivo crosslinking and mapping of photosynthetic membrane protein complexes in the chloroplasts of living Chlamydomonas reinhardtii (C. reinhardtii) cells under cultural conditions. Poly(lactic-co-glycolic acid) (PLGA) and poly(lactic-co-glycolic acid)-poly(ethylene glycol) (PLGA-PEG) nanoparticles were fabricated to deliver bis(succinimidyl)propargyl with a nitro compound (BSPNO) into the chloroplasts to crosslink photosynthetic membrane protein complexes. After the in vivo crosslinked protein complexes were extracted and digested, mass spectrometry was employed to detect lysine-specific crosslinked peptides for further elucidating the protein conformations and interactions. With this method, the weak interactions between extrinsic proteins in the luminal side (PsbL and PsbH) and the core subunits (CP47 and CP43) in photosynthetic protein complexes were directly captured in living cells. Additionally, the previously uncharacterized protein (Cre07.g335700) was bound to the light-harvesting proteins, which was related to the biosynthesis of light-harvesting antennae. These results indicated that in vivo analysis of photosynthetic protein complexes based on crosslinker nanocarriers was expected to not only figure out the difficulty in the study of photosynthetic protein complexes in living cells but also provide an approach to explore transient and weak interactions and the function of uncharacterized proteins.

Surface Nanosieving Polyether Sulfone Particles with Graphene Oxide Encapsulation for the Negative Isolation toward Extracellular Vesicles.

Extracellular vesicles (EVs) contain specific biomarkers for disease diagnosis. Current EV isolation methods are hampered in important biological applications due to their low recovery and purity. Herein, we first present a novel EV negative isolation strategy based on surface nanosieving polyether sulfone particles with graphene oxide encapsulation (SNAPs) by which the coexisting proteins are irreversibly adsorbed by graphene oxide (GO) inside the particles, while EVs with large sizes are excluded from the outside due to the well-defined surface pore sizes (10-40 nm). By this method, the purity of the isolated EVs from urine could be achieved 4.91 ± 1.01e10 particles/µg, 40.9-234 times higher than those obtained by the ultracentrifugation (UC), size-exclusion chromatography (SEC), and PEG-based precipitation. In addition, recovery ranging from 90.4 to 93.8% could be obtained with excellent reproducibility (RSD < 6%). This was 1.8-4.3 times higher than those obtained via SEC and UC, comparable to that obtained by PEG-based precipitation. Taking advantage of this strategy, we further isolated urinary EVs from IgA nephropathy (IgAN) patients and healthy donors for comparative proteome analysis, by which significantly regulated EV proteins were found to distinguish IgAN patients from healthy donors. All of the results indicated that our strategy would provide a new avenue for highly efficient EV isolation to enable many important clinical applications.

Polyethyleneimine-functionalized Fe3O4/attapulgite particles for hydrophilic interaction-based magnetic dispersive solid-phase extraction of fluoroquinolones in chicken muscle.

Fluoroquinolone (FQ) residues in foods of animal origin may threaten public health but are challenging to determine because of their low contents and complex matrices. In this study, novel polyethyleneimine-functionalized Fe3O4/attapulgite magnetic particles were prepared by a simple co-mixing method and applied as hydrophilic sorbents for the magnetic dispersive solid-phase extraction (MSPE) of three FQs, i.e., ciprofloxacin, norfloxacin, and enrofloxacin, from chicken muscle samples. The preparation of the magnetic particles was of high reproducibility and the products could be reused many times with high adsorption capacity. The key experimental factors possibly influencing the extraction efficiencies, including sample solution, extraction time, sample loading volume, desorption solution, desorption time, and elution volume were investigated. Under optimum MSPE conditions, the analytes in chicken muscle samples were extracted and then determined by RPLC-MS/MS in MRM mode. Good linearity was obtained for the analytes with correlation coefficients ranged from 0.9975 to 0.9995. The limits of detection were in the range of 0.02-0.08 µg kg-1, and the recoveries of the spiked FQs in chicken muscle samples ranged from 83.9 to 98.7% with relative standard deviations of 1.3-6.8% (n = 3). Compared with the traditional MSPE methods based on hydrophobic mechanism, this hydrophilic interaction-based method significantly simplifies the sample pretreatment procedure and improves repeatability. This method is promising for accurate monitoring of FQs in foods of animal origin.

Hydrophilic molecularly imprinted polymers functionalized magnetic carbon nanotubes for selective extraction of cyclic adenosine monophosphate from winter jujube.

In this work, a green strategy was developed to prepare molecularly imprinted polymers functionalized magnetic carbon nanotubes in aqueous phase under mild conditions for cyclic adenosine monophosphate. Thanks to water solubility of chitosan, a natural polysaccharide which is rich in amino and hydroxyl groups, provided the feasibility to synthesize the green molecularly imprinted polymers for water soluble template in aqueous media. Coupled with high-performance liquid chromatography, the method exhibited a short equilibrium time (6 min), high adsorption capacity (22.42 µg/mg), high magnetic susceptibility, and good selectivity to template molecule with the imprinting factor of 2.94. A good linearity in the range of 0.020-3.0 mg/mL for target was obtained with a correlation coefficient of 0.9998. The limit of detection (signal-to-noise ratio = 3) and limit of quantitation (signal-to-noise ratio = 10) of the magnetic solid phase extraction method for cyclic adenosine monophosphate were 5 and 15 ng/mg, respectively. And the practical application of chitosan-based molecularly imprinted polymers as adsorbent to isolate and determine cyclic adenosine monophosphate in real natural samples (winter jujube) was demonstrated.

Carbon dots-embedded epitope imprinted polymer for targeted fluorescence imaging of cervical cancer via recognition of epidermal growth factor receptor.

A carbon dots-embedded epitope imprinted polymer (C-MIP) was fabricated for targeted fluorescence imaging of cervical cancer by specifically recognizing the epidermal growth factor receptor (EGFR). The core-shell C-MIP was prepared by a reverse microemulsion polymerization method. This method used silica nanoparticles embedded with carbon dots as carriers, acrylamide as the main functional monomer, and N-terminal nonapeptides of EGFR modified by palmitic acid as templates. A series of characterizations (transmission electron microscope, dynamic light scattering, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, zeta potential, and energy dispersive X-ray spectroscopy) prove the successful synthesis of C-MIP. The fluorescence of C-MIP is quenched by the epitopes of EGFR due to the specific recognition of epitopes of EGFR through their imprinted cavities (analytical excitation/emission wavelengths, 540 nm/610 nm). The linear range of fluorescence quenching is 2.0 to 15.0 µg mL-1 and the determination limit is 0.73 µg mL-1. The targeted imaging capabilities of C-MIP are demonstrated through in vitro and in vivo experiments. The laser confocal imaging results indicate that HeLa cells (over-expression EGFR) incubated with C-MIP show stronger fluorescence than that of MCF-7 cells (low-expression EGFR), revealing that C-MIP can target tumor cells overexpressing EGFR. The results of imaging experiments in tumor-bearing mice exhibit that C-MIP has a better imaging effect than C-NIP, which further proves the targeted imaging ability of C-MIP in vivo. Graphical abstract An oriented epitope imprinted polymer embedded with carbon dots was prepared for the determination of the epitopes of epidermal growth factor receptor and targeted fluorescence imaging of cervical cancer.

Artificial Antibody with Site-Enhanced Multivalent Aptamers for Specific Capture of Circulating Tumor Cells.

Isolation of circulating tumor cells (CTCs) from blood holds great potential to diagnose cancers and discover therapeutic targets. Herein, we reported a novel kind of artificial antibody, the cell-imprinted hydrogel with site-directed modification of aptamers (APT-CIH) to achieve the specific capture of CTCs. Cell-imprinted sites not only could be used to recognize target cells but also could be used as efficient scaffolds for assembling aptamers to enhance the capture efficiency and selectivity. Due to the synergistic effect of conformation recognition and multivalent interaction between the aptamers and target cells, APT-CIH showed high capture efficiency and selectivity to SMMC-7721 cells. In the coexistence of the 1000 times leukemia Jurkat cells, the enrichment factor of APT-CIH could reach as high as 21.6 ± 3.1 toward target cells, while that relied only on cell imprinting or aptamer affinity was 8.1 ± 5.0 or 10.1 ± 1.3, respectively. Furthermore, the capture efficiency could reach 58.2% ± 10.9% with 1000 SMMC-7721 cells spiked in 1 mL of blood. Moreover, 92% of the captured cells could be released, beneficial to carry out further biological and clinical study of CTCs. These results demonstrated that APT-CIH might have great potential in CTCs analysis.

Advances in exosome isolation methods and their applications in proteomic analysis of biological samples.

Exosomes are membrane-bound vesicles secreted by cells, and contain various important biological molecules, such as lipids, proteins, messenger RNAs, microRNAs, and noncoding RNAs. Emerging evidence demonstrates that proteomic analysis of exosomes is of great significance in studying metabolic diseases, tumor metastasis, immune regulation, and so forth. However, exosome proteomic analysis has high requirements with regard to the purity of collected exosomes. Here recent advances in the methods for isolating exosomes and their applications in proteomic analysis are summarized. Graphical abstract.

3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment under Physiological Conditions.

Boronate affinity materials have been successfully used for the selective recognition of glycoproteins. However, by such materials, the large-scale glycoproteins enrichment from human plasma under physiological conditions is rarely reported. In this work, 3-carboxybenzoboroxole (CBX) functionalized polyethylenimine (PEI) modified magnetic graphene oxide nanocomposites were synthesized. Benefitting from the low pKa value of CBX (∼6.9) and PEI dendrimer-assisted multivalent binding, the Freundlich constant (KF) for the adsorption of horseradish peroxidase (HRP) was 3.0-7.3 times higher than that obtained by previous work, displaying the high enrichment capacity. Moreover, PEI could improve the hydrophilicity of nanocomposites and reduce nonglycoprotein adsorption. Therefore, such nanocomposites were successfully applied to the analysis of human plasma glycoproteome under physiological conditions, and the identified glycoproteins number and recognition selectivity was increased when compared to the results obtained by previous boronic acid-functionalized particles (Sil@Poly(APBA-co-MBAAm)) under common alkaline condition (137 vs 78 and 67.8% vs 57.8%, respectively). In addition, thrombin (F2), an important plasma glycoprotein, labile under alkaline conditions, was specifically identified by our method, demonstrating the great promise of such nanocomposites in the deep-coverage glycoproteome analysis.

Silicon nanoparticles coated with an epitope-imprinted polymer for fluorometric determination of cytochrome c.

The authors describe a composite consisting of silicon nanoparticles that were first coated with SiO2 and then with a molecularly imprinted polymer (SiNP@SiO2@MIP). The MIP was generated by dual epitope imprinting such that it can recognize cytochrome c (Cyt c). The MIP on the NPs was prepared from the functional monomer zinc(II) acrylate (ZnA), the crosslinker ethylene glycol dimethacrylate and the initiator 2,2'-azoisobutyronitrile. Dual epitope templates for Cyt c included (a) a C-terminal nonapeptide (AYLKKATNE), and (b) an N-terminal nonapeptide (GDVEKGKKI). The chelation between Zn(II) of ZnA and the amino groups or hydroxy groups of the template nonapeptides warrants good recognition and capture of Cyt c. The fluorescence originating from SiNPs has excitation/emission peaks at 360/480 nm and is quenched by Cyt c in the 0.50-40.0 µM concentration range. The correlation coefficient for the calibration plot of the imprinted NPs is 0.9937. The detection limit is 0.32 ± 0.01 µM, the precisions of six replicate detections at levels of 0.5, 20 and 40 µM Cyt c are 3.2, 2.7 and 2.8%, respectively, and the imprinting factor is 2.43. Compared to single epitope template imprinting, dual epitope imprinting results in improved selectivity. The imprinted nanoparticles can discriminate Cyt c even if one amino acid is mismatched. The method was applied to the determination of Cyt c in spiked diluted human serum and gave recoveries between 94.0 and 107.5%. Graphical Abstract A fluorescent material of the architecture silicon nanoparticle@SiO2@molecularly imprinted polymer (SiNP@SiO2@MIP) was fabricated by dual epitope imprinting and a metal-chelating method. The chelation between Zn(II) of the functional monomer zinc(II) acrylate and the amino groups or hydroxy groups of template warrants that the material recognizes and captures cytochrome c well, and this results in fluorescence quenching.

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification.

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.

Multiepitope Templates Imprinted Particles for the Simultaneous Capture of Various Target Proteins.

To achieve the simultaneous capture of various target proteins, the multiepitope templates imprinted particles were developed by phase inversion-based poly(ether sulfone) (PES) self-assembly. Herein, with the top three high-abundance proteins in the human plasma, serum albumin, immunoglobulin G, and transferrin, as the target proteins, their N-terminal peptides were synthesized as the epitope templates. After the preorganization of three epitopes and PES in dimethylacetamide, the multiepitope templates imprinted particles were formed in water through self-assembly, by which the simultaneous recognition of three target proteins in human plasma was achieved with high selectivity. Furthermore, the binding kinetics study proved that the adsorption mechanism in this imprinting system toward three epitope templates was the same as that on the single-epitope imprinting polymer. These results demonstrate that our proposed multiepitope templates imprinting strategy might open a new era of artificial antibodies to achieve the recognition of various targets simultaneously.

Effective isolation of exosomes with polyethylene glycol from cell culture supernatant for in-depth proteome profiling.

Exosomes are secreted nanovesicles shed by almost all kinds of cells. Recently, increased interest has been focused on these extracellular vesicles as natural carriers transporting biological contents for intercellular communication. However, current isolation techniques, such as ultracentrifugation, are not convenient and often require specialized equipment. Herein, we describe a polyethylene glycol (PEG)-based approach, which could permit facile, low-cost and effective isolation of exosomes from cell culture supernatant. High-resolution electron microscopes clearly visualized the size and morphology of isolated exosome aggregates, implying the mechanism of PEG-based precipitation. Combined with tandem mass spectrometry analysis, 6299 protein groups encoded by 5120 genes were successfully characterized from HeLa cell culture supernatant, including numerous exosome proteins which could overlap 97% of the Top 100 exosome marker proteins recorded in the ExoCarta database, as well as a series of low-abundance cytokines and biomarkers. Furthermore, we found a higher ratio of neo-cleavage sites in proteins identified from exosomes compared with cellular proteins, revealing the potential roles of exosomes in accumulation and transportation of protein degradation intermediates.

Ionic liquid-based zwitterionic organic polymer monolithic column for capillary hydrophilic interaction chromatography.

In the current study, a novel ionic liquid-based zwitterionic organic polymer monolithic column was developed by copolymerizing 1-vinyl-3-(butyl-4-sulfonate) imidazolium, acrylamide and N,N'-methylenebisacrylamide in a quaternary porogenic solvent consisting of formamide, dimethyl sulphoxide, polyethylene glycol 8000 and polyethylene glycol 10,000 for capillary hydrophilic interaction chromatography. The monolithic stationary phase was optimized by adjusting the amount of monomer in the polymerization solution along with the composition of porogenic solvent. The optimized monolith exhibited excellent selectivity and favorable retention for nucleosides and benzoic acid derivatives. The primary factors affecting the separation efficiency of the monolithic column (including acetonitrile content, pH, and buffer salt concentration in the mobile phase) have been thoroughly evaluated. Excellent reproducibility of the retention times for five nucleosides was achieved, with relative standard deviations of run-to-run (n = 3), column-to-column (n = 3) and batch-to-batch (n = 3) in the range of 0.18-0.48%, 2.33-4.20% and 3.07-6.50%, respectively.

Hydrophilic solid-phase extraction of melamine with ampholine-modified hybrid organic-inorganic silica material.

In this work, an ampholine-functionalized hybrid organic-inorganic silica sorbent was successfully used to extract melamine from a milk formula sample by a hydrophilic interaction solid-phase extraction protocol. Primary factors affecting the extraction efficiency of the material such as extraction solvent, elution solvent, sample loading volume, and elution volume have been thoroughly optimized. Under the optimized hydrophilic solid-phase extraction conditions, the recoveries of melamine spiked in milk formula samples ranged from 86.2 to 101.8% with relative standard deviations of 4.1-9.4% (n = 3). The limit of detection (S/N = 3) was 0.32 µg/g. The adsorption capacity toward melamine was 30 µg of melamine per grams of sorbent. Due to its simplicity, rapidity and cost effectiveness, the newly developed hydrophilic solid-phase extraction method should provide a promising tool for daily monitoring of doped melamine in milk formula.

Boronate affinity monolith with a gold nanoparticle-modified hydrophilic polymer as a matrix for the highly specific capture of glycoproteins.

As low abundance is the great obstacle for glycoprotein analysis, the development of materials with high efficiency and selectivity for glycoprotein enrichment is a prerequisite in glycoproteome research. Herein, we report a new kind of hydrophilic boronate affinity monolith by attaching 4-mercaptophenylboronic acid (MPBA) with 2-mercaptoethylamine (MPA) on the gold nanoparticle-modified poly(glycidyl methacrylate-co-poly(ethylene glycol) diacrylate)) monolith for glycoprotein enrichment. With poly(ethylene glycol) diacrylate as the cross-linker and the further modification of gold nanoparticles, the matrix has advantages of good hydrophilicity and enhanced surface area, which are beneficial to improve the enrichment selectivity and efficiency for glycoproteins. The attachment of MPBA and MPA provide intramolecular BN coordination, which could further enhance the specificity of glycoprotein capture. Such a boronate affinity monolith was applied to enrich horseradish peroxidase (HRP) from the mixture of HRP and bovine serum albumin (BSA), and high selectivity was obtained even at a mass ratio of 1:1000. In addition, the binding capacity of ovalbumin on such monolith reached 390 µg g(-1) . Furthermore, the average recovery of HRP on the prepared affinity monoliths was (84.8±1.9) %, obtained in three times enrichment with the same column. Finally, the boronate affinity monolith was successfully applied for the human-plasma glycoproteome analysis. As a result, 160 glycoproteins were credibly identified from 9 µg of human plasma, demonstrating the great potential of such a monolith for large-scale glycoproteome research.

Dual Template Molecularly Imprinted Polymers Targeting Blockade of CD47 for Enhanced Macrophage Phagocytosis and Synergistic Antimetabolic Therapy.

Glycinamide ribonucleotide formyltransferase (GARFT) is an important enzyme in the folate metabolism pathway, and chemical drugs targeting GARFT have been used in tumor treatments over the past few decades. The development of novel antimetabolism drugs that target GARFT with improved performance and superior activity remains an attractive strategy. Herein, we proposed a targeted double-template molecularly imprinted polymer (MIP) for enhancing macrophage phagocytosis and synergistic antimetabolic therapy. The double-template MIP was prepared by imprinting the exposed peptide segment of the extracellular domain of CD47 and the active center of GARFT. Owing to the imprinted cavities on the surface of MIP, it can actively target cancer cells and mask the "do not eat me" signal upon binding to CD47 thereby blocking the CD47-SIRPα pathway and ultimately enhancing phagocytosis by macrophages. In addition, MIP can specifically bind to the active center of GARFT upon entry into the cells, thereby inhibiting its catalytic activity and ultimately interfering with the normal expression of DNA. A series of cell experiments demonstrated that MIP can effectively target CD47 overexpressed 4T1 cancer cells and inhibit the growth of 4T1 cells. The enhanced phagocytosis ability of macrophages-RAW264.7 cells was also clearly observed by confocal imaging experiments. In vivo experiments also showed that the MIP exhibited a satisfactory tumor inhibition effect. Therefore, this study provides a new idea for the application of molecular imprinting technology to antimetabolic therapy in conjunction with macrophage-mediated immunotherapy.

Tracking the effects of PLGA-based nanoparticles on protein expression in living cells through quantitative proteomics.

Mass spectrometry (MS)-based proteomics can identify and quantify the differential abundance of expressed proteins in parallel, and bottom-up proteomic approaches are even approaching comprehensive coverage of the complex eukaryotic proteome. Protein-nanoparticle (NP) interactions have been extensively studied owing to their importance in biological applications and nanotoxicology. However, the proteome-level effects of NPs on cells have received little attention, although changes in protein abundance can reflect the direct effects of nanocarriers on protein expression. Herein, we investigated the effect of PLGA-based NPs on protein expression in HepG2 cells using a label-free quantitative proteomics approach with data independent acquisition (DIA). The percentage of two-fold change in the protein expression of cells treated with PLGA-based NPs was less than 10.15% during a 6 hour observation period. Among the changed proteins, we found that dynamic proteins involved in cell division, localization, and transport are more likely to be more susceptible to PLGA-based NPs.

Preparation and application of hollow molecularly imprinted polymers with a super-high selectivity to the template protein.

Protein-imprinted polymers with hollow cores that have a super-high imprinting factor were prepared by etching the core of the surface-imprinted polymers that used silica particles as the support. Lysozyme as template was modified onto the surface of silica particles by a covalent method, and after polymerization and the removal of template molecules, channels through the polymer layer were formed, which allowed a single-protein molecule to come into the hollow core and attach to the binding sites inside the polymer layer. The adsorption experiments demonstrated that the hollow imprinted polymers had an extremely high binding capacity and selectivity, and thus a super-high imprinting factor was obtained. The as-prepared imprinted polymers were used to separate the template lysozyme from egg white successfully, indicating its high selectivity and potential application in the field of separation of protein from real samples.

Screening of phage-displayed human liver cDNA library against doxorubicin with drug-immobilized monolithic polyacrylamide cryogel.

Monolithic polyacrylamide cryogel was prepared and utilized as a new matrix for drug immobilization to screen against phage-displayed human liver cDNA library. The macropores and hydrophilic nature of the cryogel made it possible for phage particles to pass unhindered. Doxorubicin, an anticancer drug, was covalently bonded to the monolithic cryogel by the glutaraldehyde method, and after five rounds of affinity selection performed in an SPE cartridge, phage clones that displayed Homo sapiens methyl CpG binding protein 2 (MeCP2 ) were selectively enriched. The interaction between doxorubicin and MeCP2 displayed phages was further validated by studying the retention of doxorubicin on MeCP2 phage-coupled cryogel. These results demonstrate that drug-coupled polyacrylamide cryogel might be a promising kind of matrix for screening target proteins against phage-displayed library.