Tandem lectin affinity chromatography monolithic columns with surface immobilised concanavalin A, wheat germ agglutinin and Ricinus communis agglutinin-I for capturing sub-glycoproteomics from breast cancer and disease-free human sera

In this study, a liquid-phase separation platform consisting of tandem lectin affinity chromatography was introduced for the selective capturing of sub-glycoproteomics that are affected in cancers, e.g. breast cancer. The platform is comprised of three monolithic columns with surface immobilised lectins including concanavalin A (Con A), wheat germ agglutinin (WGA) and Ricinus communis agglutinin-I (RCA-I). While WGA and Con A have specificities directed towards the core portion of N-glycans on the glycoprotein surface, RCA-I specifically interacts with the non-reducing terminal moieties of the outer chain structures of N-glycans. The effects of the order in which the three lectin columns were arranged in the tandem columns format were evaluated. The most suitable order proved to be WGA → Con A → RCA-I (denoted as WCR) as far as the number of captured proteins was concerned. The WCR tandem columns allowed the capture of 113 and 112 proteins from disease-free and breast cancer sera, respectively, corresponding to 75 and 65 non-redundant proteins, respectively. Using mass spectral count ratios and Q-Q plots yielded a panel of 23 non-redundant differentially expressed proteins (i.e. a panel of 23 candidate markers), which should in principle be more representative of a pathophysiological state than a single marker candidate.     

Affinity chromatography of lactate dehydrogenase and wheat germ lectin on new gels bearing carboxylic functions

The suitability of two new functionalized copolymer gels for use in affinity chromatography has been examined. Both gels were substituted by two ligands, one being specific for lactate dehydrogenase and the other for wheat germ lectin. The derivatives thus obtained were used successfully for the purification of two proteins with different biological activities.     

麦胚凝集素亲和色谱富集糖肽过程中的肽键裂解

蛋白质的糖基化是最重要的翻译后修饰之一,与蛋白质结构和功能的关系密切。凝集素亲和色谱是蛋白质糖基化研究中很常用的工具,不同的凝集素可以对不同的单糖或寡糖有特异的富集作用。麦胚凝集素（WGA）由于其特异作用的糖型广泛存在而成为使用最多的凝集素之一。在本研究中,发现将WGA用于糖肽亲和富集会导致部分肽段的降解,从而导致后续的肽段序列分析的失败。本文用4种标准蛋白质对这种现象进行了验证,结果表明肽段的降解可以发生在多个位点,其中较多地发生在酪氨酸、苯丙氨酸及亮氨酸的羧基端。这一结果提示：在糖蛋白质组研究中,如果应用WGA富集糖肽并采用质谱进行鉴定,则采用半酶切或非特异性酶切的检索策略更为合适。     

Chemometric classification of reversed-phase high-performance liquid chromatography columns

Cluster analysis and principal components analysis have been used to classify nine octadecyl (C18) high-performance liquid chromatography (HPLC) columns into three general groups displaying similar chromatographic behaviour. Principal components analysis was also able to identify the key test compounds on which the classification was most highly dependent. These identifications agreed with the classification and test compound selection by an HPLC specialist. In addition, the chemometric techniques can easily be extended to many more columns and test measurements than could be conveniently examined by a human expert.     

Rapid analysis of membrane glycopeptides by gel permeation high-performance liquid chromatography

A rapid procedure for the analysis of glycopeptides has been developed using gel permeation high-performance liquid chromatography (HPLC). Glycopeptides derived by exhaustive pronase digestion of glycoproteins from radiolabeled human tumor and normal cell lines were chromatographed on DuPont GF-250 and GF-450 gel permeation columns in buffers containing non-ionic detergents. Effective separations of glycopeptides ranging in molecular mass from less than 600 daltons to more than 20,000 daltons, equivalent to the separation range of Sephadex G50 chromatography, were achieved in 7 min. The separations were dependent upon the use of an isocratic mobile phase, that contained a low-ionic-strength Tris buffer and Nonidet P-40 or Triton X-100. The mobilities of protein standards indicated the occurrence of a biphasic elution system, which favored the separation of species with molecular masses below 20,000 daltons. Glycopeptides isolated by this method could be applied directly to lectin or ion-exchange columns or could be digested with neuraminidase, endo H or other enzymes without further treatment. Removal of sialic acid from the glycopeptides caused a dramatic increase in retention time. Using this method, glycopeptides could be isolated rapidly and in high yield. The ease, speed and reproducibility of the separations and compatibility of the solvent systems with affinity or ion-exchange chromatography techniques make this gel permeation HPLC method an ideal initial step in the purification of glycopeptides.     

High-performance affinity chromatography of human serum concanavalin A binding proteins

A high-performance concanavalin A (Con A) affinity column Gelpack GL-L55C (Hitachi Kasei Industries) was successfully used for the fractionation of human serum Con A-binding proteins. Serum proteins that have strong affinity to Con A (ca. 11% of the recovered proteins) could be fractionated within 80 min. By analysing the eluates from the column by micro two-dimensional electrophoresis, followed by blotting and Con A staining, the specificity of the column was effectively visualized. Although the protein-binding capacity of the column gradually decreased during repeated loading of serum or tissue extracts, the specificity of the column to Con A-binding proteins did not change. Serum lipoproteins have been eluted from the column with 6 M urea, suggesting that the capacity decrease is caused by the binding of lipids or lipoproteins to the column.     

Preparation of monolithic silica columns for high-performance liquid chromatography

Preparation methods of monolithic silica columns for HPLC including the surface modification were reviewed. Chemical modification methods recently reported to obtain stationary phases for reversed-phase (RP), chiral, ion-exchange, and hydrophilic interaction chromatography (HILIC) separations were discussed. Recent results related to preparation methods of monolithic silica were also covered. The characteristics and properties of silica monoliths and some applications of monolithic silica columns for different analytical and bioanalytical fields will be commented.     

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.     

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.     

Preparative high-performance liquid chromatography with reversed phase packed glass columns

An efficient system for preparative reversed-phase separations with packed glass columns is described. The advantage of this system is the use of relatively simple and inexpensive equipment. Column performance, load capacity, effect of the feed volume and the feed concentration on peak broadening are shown. The influence of the selectivity and the capacity factors on column load have been measured. The effect of the column dimensions is demonstrated by means of practical applications. The loading capacity of a column depends on the thermodynamic proporties of the separation system used. It is therefore not expedient in preparative chromatography to correlate the loading capacity of a column by means of grams dissolved per grams of adsorbent.     

Rapid purification of antisteroid antibodies by high-performance liquid affinity chromatography

An adsorbent for the high-performance affinity chromatography of antisteroid antibodies was prepared, based on a commercial pre-packed column. The column contained activated microparticulate silica beads bearing epoxide functions, on which the steroid dexamethasone was covalently linked. The column was used successfully for the rapid and complete isolation of several hundred microgram amounts of specific antidexamethasone antibodies from rabbit antisera. The practical aspects of the purification procedure, especially the optimization of the washing and of the elution steps, are detailed. Despite non-biospecific elution with 20% acetonitrile in an acidic buffer, the purification yield was very satisfactory and the biological activity of the purified immunoglobulins appeared excellent.     

Affinity chromatography of β-N-acetylhexosaminidases on columns with mixed charge and affinity functions

Analysis was made of the nature of interactions between β-N-acetylhexosaminidase and affinity chromatography gels made by coupling 2-acetamido-N-(6-aminohexanoyl)-2-deoxy-β-D-glucopyranosylamine (ANAG) to CNBr-Sepharose columns. This showed that although specific binding of the enzyme to the immobilized ligand was too weak to cause retention, compound affinity in which charge interactions were involved could be exploited for purification of the enzyme. Evidence is given for the specificity of interaction of the enzyme with immobilized ligand and for biospecific desorption of β-N-acetylhexosaminidases from ANAG-Sepharose columns. A method was developed for the purification of placental β-N-acetylhexosaminidase A in mg amounts starting from crude extracts.     

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.     

The use of microparticulate guard columns in reverse-phase high-performance liquid chromatography

Summary The use of mciroparticulate guard columns in conjunction with high efficiency reverse-phase columns has been evaluated in terms of system efficiency and peak symmetry using three standard test mixtures. The effects of linking tube dimensions, particle size of guard column packing and the use of low or zero dead volume couplings have been investigated. Both valve and stopped-flow injection techniques have been used. Recommendations are given for the most efficient use of guard columns.     

Radial heterogeneity of some analytical columns used in high-performance liquid chromatography

An on-column electrochemical microdetector was used to determine accurately the radial distribution of the mobile phase velocity and of the column efficiency at the exit of three common analytical columns, namely a 100 mm × 4.6 mm C18 bonded silica-based monolithic column, a 150 mm × 4.6 mm column packed with 2.7 μm porous shell particles of C18 bonded silica (HALO), and a 150 mm × 4.6 mm column packed with 3 μm fully porous C18 bonded silica particles (LUNA). The results obtained demonstrate that all three columns are not radially homogeneous. In all three cases, the efficiency was found to be lower in the wall region of the column than in its core region (the central core with a radius of 1/3 the column inner radius). The decrease in local efficiency from the core to the wall regions was lower in the case of the monolith (ca. 25%) than in that of the two particle-packed columns (ca. 35–50%). The mobile phase velocity was found to be ca. 1.5% higher in the wall than in the core region of the monolithic column while, in contrast, it was ca. 2.5–4.0% lower in the wall region for the two particle-packed columns.     

Fractionation of glycoprotein-derived oligosaccharides by affinity chromatography using immobilized lectin columns

Lectin affinity column chromatography is becoming a method of choice for the fractionation and purification of oligosaccharides, especially N-linked oligosaccharides. Using lectin affinity, it is easy to separate structural isomers and to isolate oligosaccharides based on specific features. Further, serial lectin column chromatography, when various lectin columns are used at the same time, can afford a very sensitive method for the fractionation and characterization of extremely small amounts of oligosaccharides. Thus, when used in conjunction with other separation techniques, lectin affinity chromatography can help to purify rapidly oligosaccharides and provide substantial information about their structural features.     

On-line coupling of sol-gel-generated immunoaffinity columns with high-performance liquid chromatography.

The paper demonstrates the possibility to use sol-gel-generated immunoaffinity columns as selective sample preparation step in on-line combination with HPLC. In the past sol-gel-generated immunoaffinity columns have only been included in off-line sample preparation schemes. Compared with conventional RP-materials on-line coupling of sol-gel-generated silica matrices with a pore structure designed to retain antibodies poses additional problems caused by their lower pressure tolerance and by the necessity to match the mobile phases not only to take into account the chromatographic properties but also the conformational stability of the antibodies. These problems have been overcome by an on-line system which can be regarded as a prototype for similar systems which exploit the selectivity of sol-gel immunoaffinity columns. The system consists of a sol-gel-generated immunoaffinity column coupled to an RP enrichment column and an analytical column. The practicality of such systems is demonstrated using the example of anti-pyrene immunoaffinity columns applied for the determination of pyrene in aqueous solutions.     

High-performance affinity chromatography of oligonucleotides on nucleic acid analogue immobilized silica gel columns.

The nucleic acid analogues poly(9-vinyladenine) (PVAd), poly(9-adenylethyl methacrylate) and poly(thymylethyl methacrylate) (PTM) were chemically bonded to porous silica gel, which had been pretreated with 3-trimethoxysilylpropyl methacrylate, by free radical copolymerization to produce novel packing materials for affinity chromatographic columns. The columns separated nucleosides and nucleotide dimers on the basis of hydrophobic interaction using an aqueous buffer and complementary hydrogen bonding interaction in methanol as an eluent. The PVAd- and PTM-silica gel columns gave a nucleobase-selective separation of oligonucleotides differing in length from mixtures of oligoadenylic and oligouridylic acids. On the PVAd-silica gel column terminal phosphate isomers of oligouridylic acid up to seven mer were resolved and the elution order of the isomers was different from that on an ODS column.     

The art and science of forming packed analytical high-performance liquid chromatography columns

Columns of packed particles still are the most popular devices for high-performance liquid chromatography (HPLC) separations because of their great utility, excellent performance and wide variety. However, the forming of packed beds for efficient, stable columns traditionally has been an art where the basics of how to form optimum beds generally was not well understood. The recent development of monolith rods was introduced in part to overcome the difficulty of producing stable beds of packing particles. However, these materials are less versatile than packed particle columns. Technology developments in recent years have produced a better understanding among those skilled in the practice of how to form optimized packed beds, and this has led to widely available, high-quality commercial columns. This presentation discusses the developments that led to the present state of column packing technology. Important steps in the packing of efficient, stable beds are described. The key step of selecting the best solvent for the slurry packing method is emphasized. Factors affecting the mechanical stability of packed columns also are discussed. The early art of packing columns now has evolved into a more scientific approach that allows the packing of good columns with a minimum of effort and time.