Preparation of a monolithic capillary column with immobilized α-mannose for affinity chromatography of lectins

A simple method for the preparation of an affinity monolithic (also called continuous bed) capillary column for α-mannose-specific lectins is described. 2-Hydroxyethyl methacrylate in combination with (+)-N,N´-diallyltartardiamide (DATD) and piperazine diacrylamide (PDA, 1,4-bisacryloyl-piperazine) as crosslinkers, were used as monomers for the monolith. After oxidation of DATD with periodate, α-mannose with spacer was bound to the aldehyde groups of the polymeric skeleton via reductive amination to form an affinity column for the separation, enrichment or binding studies of mannose-specific lectins. The permeability of the column was excellent. The porosity of the monolith was investigated by scanning electron microscope (SEM) and inverse size exclusion chromatography (ISEC). The affinity of the monolith was evaluated by frontal analysis (FA) and fluorescence microscopy (FM) using fluorescently labeled concanavalin (Con A). Frontal affinity chromatography showed a specific interaction of two different lectins with the α-mannose-modified monolith. According to FM the affinity sites were evenly distributed over the monolithic bed.     

Fe3+ immobilized metal affinity chromatography with silica monolithic capillary column for phosphoproteome analysis

Immobilized metal affinity chromatography (IMAC) is a commonly used technique for phosphoproteome analysis due to its high affinity for adsorption of phosphopeptides. Miniaturization of IMAC column is essential for the analysis of a small amount of sample. Nanoscale IMAC column was prepared by chemical modification of silica monolith with iminodiacetic acid (IDA) followed by the immobilization of Fe3+ ion inside the capillary. It was demonstrated that Fe3+-IDA silica monolithic IMAC capillary column could specifically capture the phosphopeptides from tryptic digest of alpha-casein with analysis by MALDI-TOF MS. The silica monolithic IMAC capillary column was manually coupled with nanoflow RPLC/nanospray ESI mass spectrometer (muRPLC-nanoESI MS) for phosphoproteome analysis. The system was validated by analysis of standard phosphoproteins and then it was applied to the analysis of protein phosphorylation in mouse liver lysate. Besides MS/MS spectra, MS/MS/MS spectra were also collected for neutral loss peak. After database search and manual validation with conservative criteria, 29 singly phosphorylated peptides were identified by analyzing a tryptic digest of only 12 mug mouse liver lysate. The results demonstrated that the silica monolithic IMAC capillary column coupled with muRPLC-nanoESI MS was very suitable for the phosphoproteome analysis of minute sample.     

Cell separation using cryogel-based affinity chromatography

In cell affinity chromatography, type-specific cell separation is based on the interaction between cell-surface receptors and an immobilized ligand on a stationary matrix. This protocol describes the preparation of monolithic polyacrylamide and polydimethylacrylamide cryogel affinity matrices that can be used as a generic type-specific cell separation approach. The supermacroporous monolithic cryogel has highly interconnected large pores (up to 100 μm) for convective migration of large particles such as mammalian cells. In this protocol, they are functionalized to immobilize a protein A ligand by a two-step derivatization of epoxy-containing cryogel monolith (reaction with ethylenediamine and glutaraldehyde). Target cells were labeled with specific antibodies and then they were captured in the cryogel through affinity with protein A. These specifically captured cells were recovered in high yields while retaining their viability by mechanical squeezing of the spongy and elastic cryogel matrices. The suggested cell separation protocol takes < 30 min for complete separation on a preprepared protein A-immobilized cryogel column.     

Monolithic chromatography on conjoint liquid chromatography columns for speciation of platinum-based chemotherapeutics in serum of cancer patients

BackgroundMonolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run.MethodsHere, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. Both the CLC columns constructed from affinity Protein G and weak anion exchange diethylamine (DEAE) disks, were applied for the speciation of cisplatin, oxaliplatin and carboplatin in spiked standard serum proteins, spiked human serum and serum of cancer patients. The analytical performances of the CLC columns used were evaluated by comparing their robustness, selectivity, repeatability and reproducibility. The separated serum proteins were detected on-line by ultraviolet (UV) and eluted Pt species by inductively coupled plasma mass spectrometry (ICP-MS). For accurate quantification of the separated Pt species (unbound Pt-based chemotherapeutic from species associated to transferrin (Tf), human serum albumin (HSA) and Immunoglobulin G (IgG)), post column isotope dilution (ID)-ICP-MS was used.ResultsThe data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. It also enables more effective cleaning of monolithic disks and to analyse larger series of serum samples than the high-pressure CLC column. Analyses of serum samples of cancer patients treated with cisplatin or carboplatin showed that Pt-chemotherapeutics were bound preferentially to HSA (around 80%). The portion of unbound Pt in general did not exceed 2%, up to 5% of Pt was associated with Tf and approximately 20% with IgG. Column recoveries, calculated as a ratio between the sum of concentrations of Pt species eluted and concentration of total Pt in serum samples, were close to 100%.ConclusionsLow-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.     

Online multidimensional separation with biphasic monolithic capillary column for shotgun proteome analysis

A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 microm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 microg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only approximately 900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.     

Experimental characterization and modelling of analytical monolithic column

Hydrodynamics, equilibrium and kinetics of adsorption in a silica-based monolithic column Chromolith Performance RP-18e (Merck KgaA, Germany) have been studied. The column permeability was calculated according to the Darcy law for laminar flow. The efficiency of the monolithic column was characterized through the height equivalent to a theoretical plate (HETP) for myoglobin, phenol and progesterone. The 2-D single channel mathematical model has been applied to describe the adsorption dynamics. Parabolic velocity profile, axial and radial diffusion in the monolith channel, linear driving force model for the mass transfer in the monolith channel skeleton wall and linear adsorption equilibrium were assumed. The mathematical model gives good prediction of the experimental elution peaks.     

免疫亲和层析法纯化棕尾别麻蝇(Sarcophagaperegrina)幼虫血淋巴凝集素

 报道了利用免疫亲和层析法纯化棕尾别麻蝇幼虫血淋巴凝集素的结果．哺乳动物红细胞能够特异地吸附凝集素．用兔红细胞与麻蝇幼虫血淋巴凝集素形成的复合体免疫供血家兔,得到麻蝇幼虫血淋巴凝集素的抗体．再利用抗体制备亲和吸附柱,通过免疫亲和层析一次性纯化了麻蝇幼虫血淋巴凝集素． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ结果显示,该凝集素的分子量约为７３ ｋ Ｄ．这一结果,与用对麻蝇幼虫血淋巴凝集素有抑制作用的糖蛋白—胎球蛋白和甲状腺球蛋白为配基,亲和层析纯化的结果完全相同,表明用这种免疫亲和层析法纯化凝集素是可行的．为不清楚专一性识别糖或专一性识别糖不典型,难于用普通亲和层析纯化的凝集素,提供了一种有效的纯化方法．     

Fast analysis of pravastatin in production media

High throughput methods (high performance liquid chromatography and capillary electrophoresis) were developed to determine pravastatin in production media. The analyses were performed on particle column, monolithic column and silica capillary filled with borate buffer pH 9.3 containing 20 mM SDS. All three methods successfully separate pravastatin from interfering compounds (matrix, mevastatin and 6-epi pravastatin) and runtimes are shorter than 1 min. Solvent consumptions for methods using small particle column, monolith column and MECK were 132, 510 and 1.5 mL h(-1). The most sensitive was the method using particle column (LOD was about 10(-5) mg mL(-1)), followed by the system using monolith column (LOD was 2 x 10(-4) mg mL(-1)) and the MECK method (LOD was about 0.02 mg mL(-1)).     

Egg-matrix for large-scale single-step affinity purification of plant lectins with different carbohydrate specificities

Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.     

Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles

Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 microm. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.     

Matrine-imprinted monolithic stationary phase for extraction and purification of matrine from Sophorae flavescentis Ait

A matrine-imprinted monolithic stationary phase (MIP monolith) was prepared by in situ polymerization for extraction and purification of matrine from Sophorae flavescentis Ait. Matrine was used as the template molecule, methacrylic acid as the function monomer, ethylene glycol dimethacrylate as the cross-linking agent, and toluene and dodecanol as the porogenic solvents. Scanning electron microscope study revealed that a monolithic structure with mesopores and 36 μm diameter nodules was obtained. The molecular recognition process and the effect of varying chromatographic conditions on separation were examined by high-performance liquid chromatography (HPLC). Hydrogen bonding, electrostatic, hydrophobic interactions and the molecular shape matching in MIP monolith cavities were proposed to be responsible for the recognition mechanism. The use of MIP monolith as a solid-phase extraction (SPE) sorbent for extraction and purification of matrine from S. flavescentis Ait was investigated. The extraction yield was 89.2% (for 3.0 mmol l(-1) matrine) with enrichment factor 29.     

Synthesis and evaluation of polymeric continuous bed (monolithic) reversed-phase gradient stationary phases for capillary liquid chromatography and capillary electrochromatography

There is a demand of novel high resolution separation media for separation of complex mixtures, particularly biological samples. One of the most flexible techniques for development of new separation media currently is synthesis of the continuous bed (monolithic) stationary phases. In this study the capillary format gradient stationary phases were formed using continuous bed (monolith) polymerization in situ. Different reversed-phase stationary phase gradients were tailored and their resolution using capillary liquid chromatography and capillary electrochromatography at isocratic mobile phase conditions was evaluated. It is demonstrated, that efficiency and resolution of the gradient stationary phases can be substantially increased comparing to the common (isotropic) stationary phases. The proposed formation approach of the gradient stationary phase is reproducible and compatible with the capillary format or microchip format separations. It can be easily automated for the separation optimizations or mass production of the capillary columns or chips.     

Analysis of protein phosphorylation by monolithic extraction columns based on poly(divinylbenzene) containing embedded titanium dioxide and zirconium dioxide nano-powders

The potential of an organic monolith with incorporated titanium dioxide (TiO(2)) and zirconium dioxide (ZrO(2)) nanoparticles was evaluated for the selective enrichment of phosphorylated peptides from tryptic digests. A pipette tip was fitted with a monolith based on divinylbenzene (DVB) of highly porous structure, which allows sample to pass through the monolithic bed. The enrichment of phosphopeptides was enhanced by increasing the pipetting cycles during the sample preparation and a higher recovery could be achieved with adequate buffer systems. A complete automated process was developed for enrichment of phosphopeptides leading to high reproducibility and resulting in a robust method designed to minimize analytical variance while providing high sensitivity at high sample throughput. The effect of particle size on the selectivity of phosphopeptides was investigated by comparative studies with nano- and microscale TiO(2) and ZrO(2) powders. Eleven phosphopeptides from alpha-casein digest could be recovered by an optimized mixture of microscale TiO(2)/ZrO(2) particles, whereas nine additional phosphopeptides could be retained by the same mixture of nano-structured material. When compared to conventional immobilized metal-ion affinity chromatography and commercial phosphorylation-enrichment kits, higher selectivity was observed in case of self fabricated tips. About 20 phosphopeptides could be retained from alpha-casein and five from beta-casein digests by using TiO(2) and ZrO(2) based extraction tips. Further selectivity for phosphopeptides was demonstrated by enriching a digest of in vitro phosphorylated extracellular signal regulated kinase 1 (ERK1). Two phosphorylated peptides of ERK1 could be identified by MALDI-MS/MS measurements and a following MASCOT database search.     

Hydrogel coated monoliths for enzymatic hydrolysis of penicillin G

The objective of this work was to develop a hydrogel-coated monolith for the entrapment of penicillin G acylase (E. coli, PGA). After screening of different hydrogels, chitosan was chosen as the carrier material for the preparation of monolithic biocatalysts. This protocol leads to active immobilized biocatalysts for the enzymatic hydrolysis of penicillin G (PenG). The monolithic biocatalyst was tested in a monolith loop reactor (MLR) and compared with conventional reactor systems using free PGA, and a commercially available immobilized PGA. The optimal immobilization protocol was found to be 5 g l(-1) PGA, 1% chitosan, 1.1% glutaraldehyde and pH 7. Final PGA loading on glass plates was 29 mg ml(-1) gel. For 400 cpsi monoliths, the final PGA loading on functionalized monoliths was 36 mg ml(-1) gel. The observed volumetric reaction rate in the MLR was 0.79 mol s(-1) m(-3) (monolith). Apart from an initial drop in activity due to wash out of PGA at higher ionic strength, no decrease in activity was observed after five subsequent activity test runs. The storage stability of the biocatalysts is at least a month without loss of activity. Although the monolithic biocatalyst as used in the MLR is still outperformed by the current industrial catalyst (immobilized preparation of PGA, 4.5 mol s(-1) m(-3) (catalyst)), the rate per gel volume is slightly higher for monolithic catalysts. Good activity and improved mechanical strength make the monolithic bioreactor an interesting alternative that deserves further investigation for this application. Although moderate internal diffusion limitations have been observed inside the gel beads and in the gel layer on the monolith channel, this is not the main reason for the large differences in reactor performance that were observed. The pH drop over the reactor as a result of the chosen method for pH control results in a decreased performance of both the MLR and the packed bed reactor compared to the batch system. A different reactor configuration including an optimal pH profile is required to increase the reactor performance. The monolithic stirrer reactor would be an interesting alternative to improve the performance of the monolith-PGA combination.     

Structural characterization of novel chitin-binding lectins from the genus Artocarpus and their antifungal activity

Two novel chitin-binding lectins from seeds of Artocarpus genus were described in this paper, one from A. integrifolia (jackfruit) and one from A. incisa (breadfruit). They were purified from saline crude extract of seeds using affinity chromatography on chitin column, size-exclusion chromatography and reverse-phase chromatography on the C-18 column. Both are 14 kDa proteins, made up of 3 chains linked by disulfide bonds. The partial amino acid sequences of the two lectins showed they are homologous to each other but not to other plant chitin-binding proteins. Thus, they cannot be classified in any known plant chitin-binding protein family, particularly because of their inter-chain covalent bonds. Their circular dichroism spectra and deconvolution showed a secondary structure content of beta-sheet and unordered elements. The lectins were thermally stable until 80 degrees C and structural changes were observed below pH 6. Both lectins inhibited the growth of Fusarium moniliforme and Saccharomyces cerevisiae, and presented hemagglutination activity against human and rabbit erythrocytes. These lectins were denoted jackin (from jackfruit) and frutackin (from breadfruit).     

Distribution of cell surface saccharides on pancreatic cells

We describe here a simple, general procedure for the purification of a variety of lectins, and for the preparation of lectin-ferritin conjugates of defined molar composition and binding properties to be used as probes for cell surface saccharides. The technique uses a “universal” affinity column for lectins and their conjugates, which consists of hog sulfated gastric mucin glycopeptides covalently coupled to agarose. The procedure involes: (a) purification of lectins by chromatography of aqueous extracts of seeds or other lectin-containing fluids over the affinity column, followed by desorption of the desired lectin with its hapten suge; (b) iodination of the lectin to serve as a marker during subsequent steps; (c) conjugation of lectin to ferritin with glutaraldehyde; (d) collection of active lectin-ferritin conjugates by affinity chromatography; and (e) separation of monomeric lectin-ferritin conjugates from larger aggregates and unconjugated lectin by gel chromatography. Based on radioactivity and absorbancy at 310 nm for lectin and ferritin, respectively, the conjugates consist of one to two molecules of lectin per ferrritin molecule. Binding studies of native lectins and their ferritin conjugates to dispersed pancreatic acinar cells showed that the conjugation procedure does not significantly alter either the affinity constant of the lectin for its receptor on the cell surface or the number of sites detected.     

The covalently bonded cellulose tris(3,5-dimethylphenylcarbamate) on a silica monolithic capillary column for enantioseparation in capillary electrochromatography

A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-microm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.     

High-performance affinity chromatography with immobilization of protein A and L-histidine on molded monolith

Reactive monoliths of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and L-histidine were immobilized on the monoliths directly or through a spacer arm, respectively. The properties of these two kinds of affinity columns were characterized, and the results showed that the columns with coupling of ligands by a spacer arm have some extent of non-specific adsorption for bovine serum albumin. The affinity column based on the monolithic polymer support provided us with good hydrodynamic characteristic, low flow resistance, and easy preparation. These two affinity columns were used for the purification of immunoglobulin G from human serum. The purity of the purified IgG was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The stability of the protein A affinity column was investigated, and its performance remained invariable after half a year. The effects of the nature and the pH of the buffer system on the adsorption capacity of human IgG on histidyl affinity column were also investigated. The protein A affinity column is favorable for rapid analysis of human IgG samples. In contrast, the advantages of mild elution conditions, high stability, as well as low cost provide the histidyl column further potential possibility for fast removal of IgG from human plasma in clinical applications.     

New monolith technology for automated anion-exchange purification of nucleic acids

Synthetic nucleic acid analysis often employs pellicular anion-exchange (AE) chromatography because it supports very high efficiency separations while offering means to control secondary structure, retention and resolution by readily modifiable chromatographic conditions. However, these pellicular anion-exchange (pAE) phases do not offer capacity sufficient for lab-scale oligonucleotide (ON) purification. In contrast, monolithic phases produce fast separations at capacities exceeding their pellicular counterparts, but do not exhibit capacities typical of fully porous, bead-based, anion-exchangers. In order to further increase monolith capacity and obtain the selectivity and mass transfer characteristics of pellicular phases, a surface-functionalized monolith was coated with pAE nanobeads (latexes) usually employed on the pellicular DNAPac phase. The nanobead-coated monolith exhibited chromatographic behaviors typical of polymer AE phases. Based on this observation the monolithic substrate surface porosity and latex diameters were co-optimized to produce a hybrid monolith harboring capacity similar to that of fully porous bead-based phases and peak shape approaching that of the pAE phases. We tested the hybrid monolith on a variety of previously developed pAE capabilities including control of ON selectivity, resolution of derivatized ONs, the ability to resolve RNA ONs harboring aberrant linkages at different positions in a single sequence and separation of phosphorothioate diastereoisomers. We compared the yield and purity of an 8 mg ON sample purified on both the new hybrid monolith and a benchmark AE column based on fully porous monodisperse beads. This comparison included an assessment of the relative selectivities of both columns. Finally, we demonstrated the ability to couple AE ON separations with ESI-MS using an automated desalting protocol. This protocol is also useful for preparing ONs for other assays, such as enzyme treatments, that may be sensitive to high salt levels.     

Preparation and evaluation of bovine serum albumin immobilized chiral monolithic column for affinity capillary electrochromatography

A system of capillary silica monolith with bovine serum albumin (BSA) functionalized through two approaches for affinity monolithic capillary electrochromatography (AMCEC) was developed. Covalent immobilization conditions for two different Schiff base methods, which employed 3-glycidopropyl trimethoxysilane (GPTS) and 3-aminopropyl trimethoxysilane (APTS) as starting materials, respectively, were investigated to obtain good and stable chiral separation. The BSA immobilized silica monoliths were evaluated in terms of morphology, electroosmotic flow, retention time, column efficiency and resolution of model compound (±)-tryptophan. The columns exhibited satisfactory run-to-run, column-to-column repeatability and maintained their enantioselectivity for more than 3 months. Both developed methods can baseline separate tryptophan enantiomers, whereas shorter retention time, better column efficiency, and enantiomeric recognition between two pairs of drug enantiomers (pantoprazole and atenolol) were obtained by the GPTS method.