Kaiguang Yang

Boronic Acid Functionalized Core-Shell Polymer Nanoparticles Prepared by Distillation Precipitation Polymerization for Glycopeptide Enrichment

The boronic acid-functionalized core-shell polymer nanoparticles, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@4-vinylphenylboronic acid (poly(MBA-co-MAA)@VPBA), were successfully synthesized for enriching glycosylated peptides. Such nanoparticles were composed of a hydrophilic polymer core prepared by distillation precipitation polymerization (DPP) and a boronic acid-functionalized shell designed for capturing glycopeptides. Owing to the relatively large amount of residual vinyl groups introduced by DPP on the core surface, the VPBA monomer was coated with high efficiency, working as the shell. Moreover, the overall polymerization route, especially the use of DPP, made the synthesis of nanoparticles facile and time-saving. With the poly(MBA-co-MAA)@VPBA nanoparticles, 18 glycopeptides from horseradish peroxidase (HRP) digest were captured and identified by MALDI-TOF mass spectrometric analysis, relative to eight glycopeptides enriched by using commercially available meta-aminophenylboronic acid agarose under the same conditions. When the concentration of the HRP digest was decreased to as low as 5 nmol, glycopeptides could still be selectively isolated by the prepared nanoparticles. Our results demonstrated that the synthetic poly(MBA-co-MAA)@VPBA nanoparticles might be a promising selective enrichment material for glycoproteome analysis.

Preparation of a new type of affinity materials combining metal coordination with molecular imprinting

A new approach, combining metal-coordination with molecular imprinting technology, was developed to prepare protein-affinity materials, which showed higher specific recognition ability towards the target protein than those prepared using either metal-coordination or molecular imprinting technology.

Protein-imprinted materials: rational design, application and challenges

AbstractProtein imprinting is a promising tool for generating artificial biomimetic receptors with antibody-like specific recognition sites. Recently, protein-imprinted materials, as potential antibody substitutes, have attracted much attention in many fields, for example chemical sensors, chromatographic stationary phases, and artificial enzymes, owing to their long-term storage stability, potential re-usability, resistance to harsh environment, and low cost. In this critical review, we focus our discussion on the rational preparation of protein-imprinted materials in terms of choice of template, functional monomer, crosslinker, and polymerization format. In addition, several highlighted applications of protein-imprinted materials are emphasized, not only in well-known fields but also in some unique fields, for example proteomics and tissue engineering. Finally, we propose challenges arising from the intrinsic properties of protein imprinting, for example obtaining the template, heterogeneous binding, and extrinsic competition, for example immobilized aptamers. FigureSchematic representation of fabrication and application of protein-imprinted materials

Molecularly imprinted polyethersulfone microspheres for the binding and recognition of bisphenol A

BPA-imprinted polyethersulfone (PES) microspheres for the binding and recognition of bisphenol A (BPA) were fabricated by means of a liquid-liquid phase separation technique. The imprinted novel PES microspheres had a porous structure with a skin layer, under which was followed by a finger-like structure. The recognition experiments with the BPA-imprinted microspheres were carried out by applying the microspheres to various BPA solutions. In water, high binding amounts of BPA were observed in the range of 19-42μmol/g capacity, but the recognition was low in the BPA water solution. With the increase of the concentration in BPA solution, the binding amounts and the recognition coefficient increased. However, 1,4-butylene glycol/water media showed high recognition of the imprinted microspheres with a low binding capacity of BPA. In addtion, with the increase of the BPA amounts in the PES solution used to prepare the imprinted microspheres, the specific recognition sites increased, and the recognition ability increased. Evidence revealed that microsphere recognition was effective for BPA due to the binding to specific recognition sites [S](sites). The imprinted microspheres showed the selectivity for BPA in the wine including BPA and other organic compounds. Charge transfer and special cavities could be employed to explain the mechanism.

Hydrophilic immobilized trypsin reactor with magnetic graphene oxide as support for high efficient proteome digestion

In this paper, magnetic Fe₃O₄ nanoparticles modified graphene oxide nanocomposites (GO-CO-NH-Fe₃O₄) were prepared by covalent bonding, via the reaction between the amino groups of fuctionalized Fe₃O₄ and the carboxylic groups of GO, confirmed by Fourier-transform infrared spectra, Raman spectroscopy, and transmission electron microscopy. With GO-CO-NH-Fe₃O₄ as a novel substrate, trypsin was immobilized via π-π stacking and hydrogen bonding interaction, and the binding capacity of trypsin reached as high as 0.275 mg/mg. Since GO-CO-NH-Fe₃O₄ worked as not only support for enzyme immobilization, but also as an excellent microwave irradiation absorber, the digestion efficiency could be further improved with microwave assistance. By such an immobilized enzymatic reactor (IMER), standard proteins could be efficiently digested within 15 s, with sequence coverages comparable or better than those obtained by conventional in-solution digestion (12 h). Since trypsin was immobilized under mild conditions, the enzymatic activity of IMER preserved at least for a month. In addition, due to the good hydrophilicity of GO, no peptide residue was observed in the sequent digestion of bovine serum albumin and myoglobin. To further confirm the efficiency of such an IMER for proteome analysis, it was applied to digest proteins extracted from rat liver, followed by nanoRPLC-ESI-MS/MS analysis. With only 5 min microwave-assisted digestion, in 3 parallel runs, totally 456 protein groups were identified, comparable to that obtained by 12 h in-solution digestion, indicating the great potential of IMERs with GO-CO-NH-Fe₃O₄ as the support for high throughput proteome study.

High throughput tryptic digestion via poly (acrylamide-co-methylenebisacrylamide) monolith based immobilized enzyme reactor

A poly (acrylamide-co-methylenebisacrylamide) (poly (AAm-co-MBA)) monolith was prepared by thermal polymerization in the 100 or 250 μm i.d. capillary. The monolithic support was activated by ethylenediamine followed by glutaraldehyde. Trypsin was then introduced to form an immobilized enzyme reactor (IMER). The prepared IMER showed a reliable mechanical stability and permeability (permeability constant K=2.65×10(-13) m(2)). With BSA as the model protein, efficient digestion was completed within 20s, yielding the sequence coverage of 57%, better than that obtained from the traditional in-solution digestion (42%), which took about 12h. Moreover, BSA down to femtomole was efficiently digested by the IMER and positively identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). To test the applicability of IMER for complex sample profiling, proteins extracted from Escherichia coli were digested by the IMER and further analyzed by nanoreversed phase liquid chromatography-electrospray ionization-mass spectrometry (nanoRPLC-ESI-MS/MS). In comparison to in-solution digestion, despite slightly fewer proteins were positively identified at a false discovery rate (FDR) of ∼1% (333 vs 411), the digestion time used was largely shortened (20s vs 24 h), implying superior digestion performance for the high throughput analysis of complex samples.

Macroporous molecularly imprinted monolithic polymer columns for protein recognition by liquid chromatography.

Macroporous cytochrome c (cyc)-imprinted monolithic polyarylamide columns were prepared, and applied for the template protein recognition by HPLC. With cyc (18.8 mg) as template, the imprinted monolithic materials were in situ polymerized in an HPLC column tube, with methacrylamide (450 mg), methacrylic acid (15.8 mg), piperazine diacrylamide (720 mg) and ammonium sulfate (390 mg) dissolved in 5 mL of phosphate buffer (pH 7.4), initiated by ammonium persulfate and TEMED. After the reaction, cyc was removed with acetic acid (10%, v/v) containing 10% w/v SDS. The non-imprinted monolithic column was prepared under the same procedure except without cyc. Retention of cyc and its competitive protein, lysozyme (lys), on molecular-imprinting polymer (MIP) and non-imprinted polymer columns was studied. When the pH value of mobile phase was 4.0, on MIP column, the retention factors of cyc and lys were 2.0 and 1.3, respectively. However, those on non-imprinted polymer column were very similar, both as 1.1. Even in competitive environment, a mixture of cyc and lys could be separated on MIP column under gradient elution, with resolution as 1.2. These results indicate that protein-imprinted monolithic polymer columns could offer obvious affinity and specific recognition to the template protein.

Preparation of protein imprinted materials by hierarchical imprinting techniques and application in selective depletion of albumin from human serum

Hierarchical imprinting was developed to prepare the protein imprinted materials, as the artificial antibody, for the selective depletion of HSA from the human serum proteome. Porcine serum albumin (PSA) was employed as the dummy template for the fabrication of the recognition sites. To demonstrate the advantages of the hierarchical imprinting, molecularly imprinted polymers prepared by hierarchical imprinting technique (h-MIPs) were compared with those obtained by bulk imprinting (b-MIPs), in terms of the binding capacity, adsorption kinetics, selectivity and synthesis reproducibility. The binding capacity of h-MIPs could reach 12 mg g−1. And saturation binding could be reached in less than 20 min for the h-MIPs. In the protein mixture, h-MIPs exhibit excellent selectivity for PSA, with imprinting factors as about 3.6, much higher than those for non-template proteins. For the proteomic application, the identified protein group number in serum treated by h-MIPs was increased to 422, which is 21% higher than that obtained from the original serum, meanwhile the identified protein group number for the Albumin Removal kit was only 376. The results demonstrate that protein imprinted polymers prepared by hierarchical imprinting technique, might become the artificial antibodies for the selective depletion of high abundance proteins in proteome study.

Surface-Imprinted Nanoparticles Prepared with a His-Tag-Anchored Epitope as the Template

The specific recognition of biomolecules by artificial antibodies has inspired fascination among chemists and biologists. Herein, we propose a new method to prepare epitope-oriented surface-imprinted nanoparticles with high template utilization efficiency. Using a His-tag as the anchor to facilitate the epitope immobilization/removal and the self-polymerization of dopamine to control the imprinted shell thickness, the prepared epitope-imprinted nanoparticles show specific recognition of the target protein. Moreover, with improved hydrophilicity of the His-tag-anchored epitope, this method opens up a universal route for imprinting epitopes with various polarities.

Dendrimer-grafted graphene oxide nanosheets as novel support for trypsin immobilization to achieve fast on-plate digestion of proteins

In this study, dendrimer grafted graphene oxide nanosheets (dGO) were prepared by covalent reaction. The successful synthesis of dGO was confirmed by Fourier-transform infrared spectra, Raman spectra, Thermo gravimetric analysis and Zeta potential. Taking advantages of large surface area, excellent biocompatibility and abundant functional groups, dGO provided an ideal substrate for trypsin immobilization. Trypsin-linked dGO was synthesized through covalent bonding using glutaraldehyde as coupling agents. The amount of trypsin immobilized on dGO nanosheets was calculated to be about 649 ± 20 mg/g. The activity of immobilized trypsin could be maintained for over 10 days at 4 °C. On-plate proteolysis could be performed without removing trypsin-linked dGO, because dGO did not interfere with matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry analysis. By such an immobilized enzymatic reactor, standard proteins could be efficiently digested within 15 min, with sequence coverages comparable or better than those obtained by conventional over-night in-solution digestion. Furthermore, trypsin-linked dGO showed high sensitivity when applied to trace samples analysis. All these results demonstrated that the developed dGO based enzymatic reactor might provide a promising tool for high throughput proteome identification.