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.     

Improved conditions for periodate/Schiff's base‐based fluorescent staining of glycoproteins with dansylhydrazine in SDS‐PAGE

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS‐PAGE was described. Down to 4–8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16‐fold higher than that of original protocol, but similar to that of Pro‐Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC‐MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel‐separated glycoproteins.     

Synthesis of Glc1Man9‐Glycoprotein Probes by a Misfolding/Enzymatic Glucosylation/Misfolding Sequence

Glycoproteins in non‐native conformations are often toxic to cells and may cause diseases, thus the quality control (QC) system eliminates these unwanted species. Lectin chaperone calreticulin and glucosidase II, both of which recognize the Glc₁Man₉ oligosaccharide on glycoproteins, are important components of the glycoprotein QC system. Reported herein is the preparation of Glc₁Man₉‐glycoproteins in both native and non‐native conformations by using the following sequence: misfolding of chemically synthesized Man₉‐glycoprotein, enzymatic glucosylation, and another misfolding step. By using synthetic glycoprotein probes, calreticulin was found to bind preferentially to a hydrophobic non‐native glycoprotein whereas glucosidase II activity was not affected by glycoprotein conformation. The results demonstrate the ability of chemical synthesis to deliver homogeneous glycoproteins in several non‐native conformations for probing the glycoprotein QC system.     

水溶性硼酸亲和富集材料的制备及基于酶联免疫吸附法检测人肝微粒体糖蛋白的应用

生物样品中的糖蛋白丰度低,且在检测中易受到其它非糖蛋白的抑制和干扰,需在分析检测前对糖蛋白进行富集,但常规的基于固相材料的糖蛋白富集方法不易与生物技术中最经典的酶联免疫吸附法(ELISA)兼容.本研究以树枝状聚合物PAMAM 4.0为载体, 结合硼酸亲和技术,制备了新型水溶性硼酸亲和富集材料(DBC),并将其应用于基于ELISA的人肝微粒体中糖蛋白的检测.采用标准糖蛋白对DBC富集条件进行优化,然后考察其灵敏度和抗干扰能力,将优化后的方法应用于复杂样品人肝微粒体糖蛋白富集.结果表明,DBC对糖蛋白的富集选择性可高达100000倍,可将糖蛋白的富集信号提高100倍.以DBC为富集材料,与ELISA分析技术相结合,只需一步简单的孵育,即可实现生物样品中糖蛋白的高灵敏度、高选择性检测,为疾病相关的糖蛋白组学研究提供了一种有效的检测手段.     

Folding of Synthetic Homogeneous Glycoproteins in the Presence of a Glycoprotein Folding Sensor Enzyme

UDP‐glucose:glycoprotein glucosyltransferase (UGGT) plays a key role in recognizing folded and misfolded glycoproteins in the glycoprotein quality control system of the endoplasmic reticulum. UGGT detects misfolded glycoproteins and re‐glucosylates them as a tag for misfolded glycoproteins. A flexible model to reproduce in vitro folding of a glycoprotein in the presence of UGGT in a mixture containing correctly folded, folding intermediates, and misfolded glycoproteins is described. The data demonstrates that UGGT can re‐glucosylate all intermediates in the in vitro folding experiments, thus indicating that UGGT inspects not only final folded products, but also the glycoprotein folding intermediates.     

Boronate affinity monolithic column incorporated with graphene oxide for the in‐tube solid‐phase microextraction of glycoproteins

A poly(vinylphenylboronic acid–ethylene glycol dimethacrylate) monolithic material incorporated with graphene oxide was synthesized inside a poly(ether ether ketone) tube. This tube with boronate affinity monolith was coupled with a high‐performance liquid chromatography system through a six‐port valve to construct an online solid‐phase microextraction with high‐performance liquid chromatography system. The performance of this solid‐phase microextraction with high‐performance liquid chromatography system was demonstrated by standard glycoprotein in aqueous samples, namely, horseradish peroxidase. Some parameters that affect the extraction performance were investigated, including sampling rate, pH of sample solution, and sampling volume. Under the optimized conditions, the developed method showed high extraction efficiency toward horseradish peroxidase. The addition of graphene oxide greatly increased the extraction efficiency of boronate affinity monolith for horseradish peroxidase. The limit of detection of the proposed method was as low as 0.01 μg/mL by using ultraviolet detection. The recognition specificity was also evaluated by analyzing the mixture of bovine serum albumin (nonglycoprotein) and horseradish peroxidase. The results showed that this material could selectively extract horseradish peroxidase from the mixture, indicating its good specificity toward glycoproteins. The proposed method was further applied for analyzing rat plasma samples spiked with horseradish peroxidase. Good recovery and repeatability were obtained.     

Novel functionalized poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres for the solid‐phase extraction of glycopeptides/glycoproteins

Novel 3‐aminophenylboronic acid functionalized poly(glycidyl methacrylate‐co‐ethylene dimethacrylate) microspheres were prepared for the solid‐phase extraction of glycopeptides/glycoproteins. The adsorption efficiency, maximum adsorption capacity, and specific recognition of the microspheres to glycoprotein were investigated. The results indicated excellent adsorption of glycoproteins by the microspheres, which are attributed to the well‐defined boronic acid brushes on the microsphere surfaces. Furthermore, a solid‐phase extraction microcolumn filled with the microspheres was used to efficiently enrich glycopeptides from enzymatic hydrolysates from human serum samples. The mass spectrometry results demonstrated that the method is suitable for the separation and enrichment of glycopeptides/glycoproteins from complex biological samples.     

Selection of phage-displayed antibodies with high affinity and specificity by electrophoresis in microfluidic devices

A method development aimed for high-throughput and automated antibody screening holds great potential for areas ranging from fundamental molecular interactions to the discovery of novel disease markers, therapeutic targets, and monoclonal antibody engineering. Surface display techniques enable efficient manipulation of large molecular libraries in small volumes. Specifically, phage display appeared as a powerful technology for selecting peptides and proteins with enhanced, target-specific binding affinities. Here, we present a phage-selection microfluidic device wherein electrophoresis was performed under two orthogonal electric fields through an agarose gel functionalized with the respective antigen. This microdevice was capable of screening and sorting in a single round high-affinity phage-displayed antibodies against virus glycoproteins, including human immunodeficiency virus-1 glycoprotein 120 or the Ebola virus glycoprotein (EBOV-GP). Phages were differentially and laterally swept depending on their antigen affinity; the high-affinity phages were recovered at channels proximal to the application site, whereas low-affinity phages migrated distal after electrophoresis. These experiments proved that the microfluidic device specifically designed for phage-selection is rapid, sensitive, and effective. Therefore, this is an efficient and cost-effective method that allowed highly controlled assay conditions for isolating and sorting high-affinity ligands displayed in phages.     

Surface plasmon resonance for real-time study of lectin-carbohydrate interactions for the differentiation and identification of glycoproteins

A study of specific interactions between lectins and glycoproteins has been carried out using surface plasmon resonance (SPR) in a flow-injection mode. Lectins were covalently immobilised on the surfaces of the microfluidic sensor chip via amine coupling and serum glycoproteins were injected into the flow channels. Specific lectin-glycoprotein interactions caused the shift of refractive index proportional to the mass concentration accumulated on the channel surface. Lectins showed different affinity to the tested glycoproteins and each glycoprotein displayed its own lectin-binding pattern. It is possible to distinguish and identify even glycoproteins with similar sugar structures by simple and quick screening. The working conditions of the assay were optimised. The lectin-based SPR made it possible to carry out the label-free detection of glycoproteins within a broad concentration range with a good linearity. Regeneration conditions for the surface of the sensor chip were found and optimised. Combination of 10 mM HCl and 10 mM glycine-HCl (pH 2.5) removes the bound glycoproteins from the lectin surface without damaging it. The kinetic and affinity parameters of lectin-glycoprotein binding were evaluated. The proposed method was tested on human glycosylated serum. Combination of the lectin panel with SPR is suitable both for specific screening and for sensitive assay of serum glycoproteins.     

Preparation and evaluation of a phenylboronate affinity monolith for selective capture of glycoproteins by capillary liquid chromatography

A phenylboronate affinity monolith was prepared and applied to the selective capture of glycoproteins from unfractionated protein mixtures. The monolith was synthesized in a 100 μm i.d capillary by an in situ polymerization procedure using a pre-polymerization mixture consisting of 4-vinylphenylboronic acid (VPBA) as functional monomer, ethylene dimethacrylate (EDMA) as crosslinker, diethylene glycol and ethylene glycol as binary porogenic solvents, and azobisisobutyronitrile (AIBN) as initiator. The prepared monolith was characterized in terms of the morphology, pore property, and recognition property. The selectivity and dynamic binding capacity were evaluated by using standard glycoproteins and nonglycoproteins as model proteins. The chromatographic results demonstrated that the phenylboronate affinity monolith had higher selectivity and binding capacity for glycoprotein than nonglycoprotein. The resulting phenylboronate affinity monolith was used as the sorbent for in-tube solid phase microextraction (in-tube SPME), and the extraction performance of the monolith was assessed by capture of ovalbumin from egg white sample.     

Affinity monolithic capillary columns for glycomics/proteomics: 1. Polymethacrylate monoliths with immobilized lectins for glycoprotein separation by affinity capillary electrochromatography and affinity nano-liquid chromatography in either a single column or columns coupled in series

In this report, microcolumn separation schemes involving monolithic capillary columns with immobilized lectins, and relevant to nanoglycomics/nanoproteomics were introduced. Positive and neutral monoliths based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) were designed for achieving lectin affinity chromatography (LAC) by nano-LC and CEC. The positive monoliths (i.e., monoliths with cationic sites) afforded relatively high permeability in nano-LC but lack predictable EOF magnitude and direction, while neutral monoliths provided a good compromise between reasonable permeability in nano-LC and predictable EOF in CEC. Lectin affinity nano-LC permitted the enrichment of classes of different glycoproteins having similar N-glycans recognized by the immobilized lectin, whereas lectin affinity CEC provided the simultaneous capturing and separation of different glycoproteins due to differences in charge-to-mass ratio. Also, this investigation demonstrated for the first time the coupling of lectin capillary columns in series (i.e., tandem columns) for enhanced separation of glycoproteins by LAC using the CEC modality. Furthermore, in the coupled columns format, glycoforms of a given glycoprotein were readily separated.     

Boronic acid-containing carbon dots array for sensitive identification of glycoproteins and cancer cells

Discrimination of glycoproteins and cell types is a significant but difficult issue. Herein, we presented a novel fluorescence sensor array for the detection and identification of glycoproteins and cancer cells based on the specific affinity between boronic acid-containing carbon dots (BA-CDs) and cis-diol residues of polysaccharides. The differential binding affinity of three BA-CDs to various glycoproteins resulted in a different fluorescence turn-on signal pattern caused by aggregation-enhanced emission (AEE), along with negligible response from other proteins. Therefore, BA-CDs encompassing sensing elements and signal indicator into one can enable a fast and accurate discrimination of glycoproteins with simple and easy operation. Seven glycoproteins could be well discriminated at a very low concentration of 10 nmol/L. The discriminating capability of glycoproteins is not sacrificed in both human urine and serum. Notably, different glycoprotein compositions of cancer cells provide more recognizable features for identification of cancer cells, comparing to the total protein. Five cell types could be identified in 15 min at a low concentration of 1000 cells/mL. This method is fast, accurate, and easy operation, and has a potential application in cancer diagnosis.     

Preparation of a boronate-functionalized affinity hybrid monolith for specific capture of glycoproteins

A novel strategy for preparation of a boronate affinity hybrid monolith was developed using a Cu(I)-catalyzed 1,3-dipolar azide–alkyne cycloaddition (CuAAC) reaction of an alkyne–boronate ligand with an azide-functionalized monolithic intermediate. An azide-functionalized hybrid monolith was first synthesized via a single-step procedure to provide reactive sites for click chemistry; then the alkyne–boronate ligands were covalently immobilized on the azide-functionalized hybrid monolith via an in-column CuAAC reaction to form a boronate affinity hybrid monolith under mild conditions. The boronate affinity monolith was characterized and evaluated by means of elemental analysis, Fourier transform infrared spectroscopy, and scanning electron microscopy. The boronate affinity hybrid monolith exhibited excellent specificity toward nucleosides and glycoproteins, which were chosen as test cis-diol-containing compounds under neutral conditions. The binding capacity of the monolith for the glycoprotein ovalbumin was 2.36 mg?·?g^-1 at pH 7.0. The practicability of the boronate affinity hybrid monolithic material was demonstrated by specific capture of the glycoproteins ovalbumin and ovotransferrin from an egg sample.

Monitoring glycosylation pattern changes of glycoproteins using multi-lectin affinity chromatography

Previously, we reported that the distribution of glycoproteins into the lectin displacement fractions of a multi-lectin affinity column was determined by the glycosylation patterns of the proteins. This distribution was observed by the sequential use of displacers specific to the lectins in the column. In this study we have evaluated the multi-lectin column (containing Concanavalin A, Wheat germ agglutinin and Jacalin lectin) to screen glycoproteins with known glycosylation pattern changes. The presence of a glycoprotein in a given displacer fraction was determined by LC-MS/MS analysis of a tryptic digest. We have used the enzyme neuraminidase to modify the oligosaccharide chains present in human transferrin, and used the enzymes, neuraminidase and fucosidase, to modify glycoproteins present in human serum. Then, by comparison with the untreated samples, we demonstrated a distribution shift of the enzyme-treated serum glycoproteins in the displacement fractions isolated from the multi-lectin column. The fractions were analyzed by a protein assay, Sequest rank comparison and peak area measurement from the extracted ion chromatogram. The results indicated that the multi-lectin affinity column (M-LAC) is sensitive to changes in the content of sialic acid and fucosyl residues present in serum glycoproteins, and has the potential to be used to screen serum proteins for glycosylation changes due to disease. In addition, the use of a glycosidase to induce specific structural changes in glycoproteins can support the development of multi-lectin column formats specific for detecting changes in the glycoproteome of certain diagnostic fluids and types of disease.     

Poly(amidoamine) dendrimer‐coated magnetic nanoparticles for the fast purification and selective enrichment of glycopeptides and glycans

Glycosylated proteins modulate various important functions of organisms. To reveal the functions of glycoproteins, in‐depth characterization studies are necessary. Although mass spectrometry is a very efficient tool for glycoproteomic and glycomic studies, efficient sample preparation methods are required prior to analyses. In the study, poly(amidoamine) dendrimer‐coated magnetic nanoparticles were presented for the specific enrichment and fast purification of glycopeptides and glycans. The enrichment and purification performance of the developed method was evaluated both at the glycopeptide, and the glycan level using several standard glycoprotein digests and released glycan samples. The poly(amidoamine) dendrimer‐coated magnetic nanoparticles not only showed selective affinity (Immunoglobulin G/Bovine Serum Albumin, 1/10 by weight) to glycopeptides and released glycans but also good sensitivity (0.4 ng/µL for Immunoglobulin G) for glycoproteomic and glycomic applications. Thirty‐five glycopeptides of Immunoglobulin G were detected after enrichment with poly(amidoamine) dendrimer‐coated magnetic nanoparticles. In addition, 55 ¹⁸O tagged deamidated glycopeptides belonging to human plasma glycoproteome were confirmed. Finally, fifty 2‐aminobenzoic acid, and 30 procainamide‐labelled human plasma N‐glycans released from human plasma glycoproteins were determined after purifications. The results indicate that the proposed enrichment and purification method using poly(amidoamine) dendrimer‐coated magnetic nanoparticles could be simply adjusted to sample preparation methods.     

Boronic acid functionalized Fe3O4 magnetic microspheres for the specific enrichment of glycoproteins

Glycoproteins are useful biomarkers and therapeutic targets for a number of diseases, including infections and cancer. However, identification and isolation of low‐abundant glycoproteins remains a significant challenge that limits their application. Thus, methods of specific and selective glycoprotein enrichment are required. In this study, novel phenylboronic acid functionalized magnetic microspheres were successfully synthesized. Fe₃O₄ microspheres were synthesized by using a hydrothermal method and were coated with tetraethyl orthosilicate using an ultrasonic method to form a core‐shell structure. Compared to the conventional mechanical stirring for 12 h, the ultrasonic method saved about 7 h in processing time, and the home‐made magnetic microspheres had better dispersibility and homogeneity. Subsequently, the magnetic microspheres were modified by addition of an amino group and a carboxyl group, in sequence. Finally, 3‐aminophenylboronic acid, as the functional monomer, was linked to the magnetic microspheres for capturing glycoprotein/glycopeptides. The results of this study indicate that phenylboronic acid functionalized magnetic microspheres show excellent adsorption performance toward glycoprotein/glycopeptides. The maximum absorbing capacity of the microspheres for fetuin was 108 mg/g, and the enrichment efficiency reached 89.7%, indicating their potential to separate and enrich glycoproteins from the complex biological samples.     

A simple,time‐saving,microwave‐assisted periodic acid–Schiff´s staining of glycoproteins on 1D electrophoretic gels

We introduce an optimized periodic acid–Schiff´s staining of glycoproteins on 1D electrophoretic gels. Thanks to heating in a household microwave oven the protocol of standard periodic acid–Schiff´s staining has been accelerated from 6 h to below 10 min employing standard chemistry. At the same time, we show that the microwave‐assisted glycoprotein staining is at least as sensitive as the conventional approach. All glycoproteins stained by the microwave‐accelerated procedure were successfully identified using MALDI TOF/TOF mass spectrometry. The ensuing reduction in gel staining time and simplification of the staining protocol should significantly increase laboratory throughput when glycoprotein detection on electrophoretic gels is required in large numbers.     

Photopolymerized sol–gel monoliths for separations of glycosylated proteins and peptides in microfluidic chips

Photopolymerized silica sol–gel monoliths, functionalized with boronic acid ligands, have been developed for protein and peptide separations in polydimethylsiloxane microfluidic devices. Pore size characterization of the monoliths was carried out with SEM, image analysis, and differential scanning calorimetry to evaluate both the micron‐sized macropores and the nanometer‐sized mesopores. Monoliths were functionalized with boronic acid using three different immobilization techniques. Batch experiments were conducted to determine the capacity of the monoliths and selectivity toward cis‐diol‐containing compounds. Conalbumin was used as a model glycoprotein, and a tryptic digest of the glycoprotein horseradish peroxidase was used as a peptide mixture to demonstrate proof‐of‐concept extraction of glycoproteins and glycopeptides by the monoliths formulated in polydimethylsiloxane microfluidic chips. For proteins, fluorescence detection was used, whereas the peptide separations employed off‐line analysis using MALDI‐MS.     

伴刀豆凝集素修饰磁性纳米粒子富集人血清中糖蛋白及质谱鉴定

发展了一种新型的磁性纳米粒子应用于人血清中特异性糖蛋白的亲和富集。制备的磁性纳米粒子具有核/壳/壳结构,即由Fe₃O₄磁性粒子/硅胶层/有机聚合物外层构成。伴刀豆凝集素A（Con A）以共价键合的形式通过短链聚乙二醇固定在粒子表面,实现了人血清中特异性糖蛋白的高效富集。富集的蛋白经过胰蛋白酶酶解后,所得的肽段经离线的二维色谱分离,用高分辨质谱共鉴定出80种蛋白。通过NetNGlyc等搜索软件分析确定其中76种为糖蛋白,分析发现在血清中质量浓度仅为0.00001 g/L的 β -2-glycoprotein 1也得到了鉴定,表明我们发展的磁性纳米粒子与凝集素相结合的方式,可以高效地富集复杂体系中与主要蛋白成分含量相差12个数量级的低丰度糖蛋白。     

Borated Titania,a New Option for the Selective Enrichment of cis‐Diol Biomolecules

As low abundance cis‐diol biomolecules are of great significance in biological organisms, preparation of materials for the selective enrichment of such compounds is highly favorable for the development of the related proteomics and metabolomics. To this end, we have prepared monolithic borated titania by a non‐aqueous sol‐gel strategy as a new inorganic affinity material for the specific capture of nucleosides, glycopeptides and glycoproteins. Benefiting from the inorganic framework, this material prevented the hydrophobic interference, which was somewhat inevitable for the mainstream organic‐based boronate affinity materials. The prepared material was carefully characterized by scanning electron microscope (SEM), X‐ray diffraction (XRD), X‐ray photoelectron spectroscopy (XPS) and nitrogen‐sorption experiments to investigate the morphology and elemental composition. The excellent performance of borated titania on enrichment of cis‐diol biomolecules was demonstrated by extracting the glycopeptides from horseradish peroxidase (HRP) digestion, standard glycoproteins, and nucleosides from a human‐urine matrix. This kind of inorganic affinity material offers a new option for selective enrichment or separation of cis‐diol biomolecules.