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.     

Approach to the comprehensive analysis of glycoproteins isolated from human serum using a multi-lectin affinity column

Glycosylation is one the most common post-translational modifications (PTM) and glycoproteins play fundamental roles in a diversity of biological processes. The development of an analytical approach to the study of variation of glycosylation patterns in serum samples has been hindered by the structural heterogeneity of this post-translational modification and the complexity of serum proteome. We have used the ability of different lectins to recognize specific glycosylation motifs to develop a specific affinity system that can achieve a comprehensive capture of serum glycoproteins. In a preliminary investigation, we evaluated the ability of five commonly used immobilized lectins to capture glycoproteins from human serum. SDS-PAGE analysis showed each lectin was able to enrich a subset of the serum glycoproteome and overlaps in lectin specificity were indeed observed. Based on these results and with the goal of studying the extent of the human serum glycoproteome, we then developed a multi-lectin affinity column containing Concanavalin A (Con A), Wheat germ and Jacalin lectin. The selection of lectins was also based on the known N-linked and O-linked glycan structures that are considered representative of the serum proteome. We then demonstrated that the capture of glycoproteins was specific, efficient and reproducible with this multi-lectin column. The results obtained with this affinity step indicated that about 10% of human serum proteins are glycosylated (weight/weight) and, after removal of six high abundance proteins, including albumin, at least 50% of the remaining proteins were glycosylated. We then evaluated the use of this affinity column to monitor changes in the pattern of glycosylation in serum samples by a controlled, stepwise release of the captured proteins from the multi-lectin affinity column with specific displacers.     

Preparation of anhydrotrypsin-immobilized diol silica as a selective adsorbent for high-performance affinity chromatography of peptides containing arginine or lysine at their C-termini

Summary Anhydrotrypsin (AHT), a catalytically inert derivative of trypsin in which the active site serine residue was converted to dehydroalanine residue by chemical modification, was immobilized onto diol silica through the activation with trifluoroethanesulfonyl chloride, and an AHT-diol-silica column was used for high-performance affinity chromatography separation of peptides containing arginine or lysine at their C-termini from the others. Improved separation in terms of speed was accomplished.     

Preparation of β-cyclodextrin sulfate-immobilized hydrophilic vinyl-polymer gel as a selective,high recovery and stable adsorbent for high-performance affinity chromatography of heparin-binding substances

Summary β-Cyclodextrin sulfate having heparin-mimicking activity was immobilized on a hydrophilic vinyl-polymer gel, TSKgel AF-Epoxy, TOYOPEARL 650M. A column packed with this material could be used for high-performance affinity chromatographic separation of heparin-binding substances such as growth factors, enzymes, coagulation factors and lipoproteins. The substances retained on the column were recovered more easily than those on a heparin-immobilized column. Furthermore, the β-Cyclodextin sulfate-immobilized gel was superior in stability to the heparin-immobilized gel when exposed to acidic and basic solutions.     

Determination of teicoplanin in human plasma and urine by affinity and reversed-phase high-performance liquid chromatography

A sensitive, highly selective and simple high-performance liquid chromatographic method for the determination of teicoplanin, a novel glycopeptide antibiotic, composed of six components, in human plasma and urine is described. After an isolation step by affinity chromatography, the antibiotic substances were chromatographed on a Nucleosil C18 column with phosphate buffer-acetonitrile according to a gradient profile. All the components were detected by their UV absorption at 240 nm. The concentration of teicoplanin was determined by using the external standard procedure. This method was applied to the sum of the six major components as well as to each of them separately. The linearity of the method was checked between 0.5 and 50 micrograms/ml for plasma and between 2 and 50 micrograms/ml for urine. The limit of detection was 0.1 microgram/ml for both biological fluids. The coefficients of variation of the between-day assays did not exceed 8.6 and 8.9% in plasma and urine, respectively. The application of the method to a pharmacokinetic study of teicoplanin after a single intravenous therapeutic dose in a patient is reported. This rapid technique also appears to be suitable for drug monitoring.     

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.     

Determination of a potential anxiolytic drug (CGS 20625) in human plasma by high-performance liquid chromatography.

An analytical method employing reversed-phase high-performance liquid chromatography is described for the determination of a potential anxiolytic agent in human plasma. This experimental drug candidate has potent and selective affinity for the central benzodiazepine receptor complex. The compound and internal standard are extracted from buffered plasma (pH 9.0) into ethyl acetate. The solvent is evaporated and the residue is reconstituted in chromatographic mobile phase. Separation is achieved on a 5-microns phenyl column with ultraviolet absorbance detection of the drug and internal standard at 270 nm. Recovery and reproducibility assessments indicate good accuracy (overall relative recovery of 101%) and precision (coefficients of variation from 2.0 to 11%) over the concentration range 10-1000 ng/ml. The limit of quantification for the method is 10 ng/ml. The method is suitable for pharmacokinetic analysis following the administration of 80 mg of drug to normal volunteers.     

A two step fractionation approach for plasma proteomics using immunodepletion of abundant proteins and multi-lectin affinity chromatography: Application to the analysis of obesity, diabetes, and hypertension diseases

Plasma is an important biological material for biomarker discovery. However, the wide dynamic range in protein concentration remains a major challenge. In this paper, we introduce the development of a proteomic platform for analysis of plasma samples. The method utilizes a double fractionation approach which combines the MARS immunodepletion column with multi-lectin affinity chromatography, M-LAC, to deplete the most abundant proteins in plasma, the majority of which are glycosylated. To determine the suitability of this methodology, we applied the workflow described in this study to a sample set composed of four groups: a control pool and three different disease pools: obesity, diabetes, and hypertension. We were able to identify changes in the level of several proteins; for example, a protein such as angiotensinogen was found to be present at high levels in patients with obesity plus diabetes and hypertension. On the other hand, apolipoprotein CI was shown to be elevated in all disease groups. A review of the literature supported our observation. The methodology presented in this report was shown to be effective for profiling changes in the plasma proteome of subjects with obesity and its associated complications such as diabetes and hypertension.     

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.

Sequence-specific DNA affinity chromatography: Application of a group-specific adsorbent for the isolation of restriction endonucleases

The use of sequence-specific DNA affinity adsorbents for the isolation of restriction endonucleases EcoRI and SphI to near homogeneity has been reported. However, the high cost of these adsorbents is a limiting factor for their wider application. This paper reports the application of sequence-specific DNA affinity ligands containing recognition sequences for 34 restriction endonucleasesas group-specific ligands in the isolation of restriction endonucleases. Crude samples of six restriction endonucleases, namely BshFI, BamHI, SmaI, SacII, PvuII and SalI, were shown to bind to these adsorbents and could be eluted at different KCl concentrations. High purification factors and recoveries were obtained. Restriction endonuclease BshFI, an isoschizomer of HaeIII, from the microorganism Bacillus sphaericus was purified to near homogeneity employing a two-step procedure which involves DNA-cellulose chromatography and oligonucleotide- ligand affinity chromatography. The enzyme exists as a monomer with an apparent relative molecular mass of 34 000 as determined by both sodium dodecyl sulphate-polyacrylamide gel electrophoresis and size-exclusion chromatography.     

Fractionation of glycoproteins according to lectin affinity and molecular size using a high-performance liquid chromatography system with sequentially coupled columns

The lectin phytohaemagglutinin was coupled to porous silica (10 micron) and used as adsorbent in a high-performance liquid affinity column sequentially coupled to a TSK-G 3000SW gel permeation column. This system was used for high-speed separation/analysis of human serum glycoproteins according to their lectin affinity and molecular size. Serum samples could be resolved in at least six different peaks representing glycoproteins exhibiting different molecular weights but with a carbohydrate content compatible with the specificity of phytohaemagglutinin.     

Amino-functionalized monolithic spin-type columns for high-throughput lectin affinity chromatography of glycoproteins

Hydrophilic poly(ethylene glycol)-based monoliths were synthesized in the spin-tip format for high-throughput applications via pulsed electron beam irradiation. Monoliths with a homogeneous porous structure and a total porosity of 69% were obtained. The cross-linked polymeric structure was further mechanically stabilized via the incorporation of silica nanoparticles. Amino-functionalization of the monoliths was accomplished by a straightforward, water-based, one-step approach that entailed the electron-beam irradiation-induced grafting of poly(allylamine). The amine functionalized spin columns showed very low unspecific protein adsorption and were successfully applied as adsorbents in lectin affinity chromatography for the purification of ovalbumin. The novel columns also outperformed a commercially available system.     

Composite cryogel with immobilized concanavalin A for affinity chromatography of glycoproteins

Composite cryogels containing porous adsorbent particles were prepared under cryogelation conditions. The composites with immobilized concanavalin A (Con A) were used for capturing glycoproteins. Adsorbent particles were introduced into the structure in order to improve the capacity and to facilitate the handling of the particles. The monolithic composite cryogels were produced from suspensions of polyvinyl alcohol particles and porous adsorbent particles and cross‐linked under acidic conditions at sub‐zero temperature. The cryogels were epoxy activated and Con A was immobilized as an affinity ligand. Binding and elution of horseradish peroxidase (HRP) was studied in batch experiment and in a chromatographic setup. Increasing adsorbent concentration in composite cryogels will increase ligand density, which therefore enhances the amount of bound HRP from 0.98 till 2.9 (milligram enzyme per milliliter of gel) in the chromatographic system. The material was evaluated in 10 cycles for binding and elution of HRP.