Combining Lectin Microcolumns with High-Resolution Separation Techniques for Enrichment of Glycoproteins and Glycopeptides

Abstract

Silica-based lectin microcolumns are described in this study together with the chemical procedures necessary for their preparation. The analytical merits of Canavalia ensiformis and Sambucus nigra lectins, [immobilized on activated macroporous silica], such as binding capacity, trapping reproducibility, and substrate selectivity, have been evaluated using model glycoproteins. The described microcolumns are applicable to high-pressure analytical schemes utilizing microvalving procedures, washing steps, and quantitative desorption for LC/MS analysis. The described analytical systems are amenable to the applications aiming at fractionation of complex glycopeptide mixtures and determination of the sites of glycosylation.High-performance affinity chromatography (HPAC) offers a number of advantages 1-4 over the classical and commonly used chromatographic technique which utilizes various immobilized ligands to retain selectively an analyte or a group of analytes. These advantages primarily include speed, ease of automation, and the application versatility. During the past decade, HPAC has been employed in biomolecular purification and enrichment and chiral separations, as well as in the study of biological interactions. 5-7 The success of any affinity chromatography, including HPAC, is largely dependent on chemical procedures employed in the attachment of a small-molecule ligand or an interacting biopolymer to a respective chromatographic support. 8-10 Their immobilization should not interfere with the native state of a ligand, facilitating access of analyte molecules and proper interactions.Lectins comprise a group of proteins with unique affinities toward carbohydrate structures. They have long been used in biomolecular isolation, carbohydrate chemistry, and histochemistry, and more recently as the mimicking agents in intercellular interaction studies. For a number of years, lectins have been utilized in various glycoprotein isolation efforts, 11-14 albeit few addressed the quantitative aspects of this approach. 15,16 Lectins with different specificities toward oligosaccharides have been immobilized to agarose and other conventional biochemical matrixes. 11-14 Some of these column materials have been commercialized. In addition, lectins have been immobilized to magnetic beads, 17 gold foils, 18 silica materials, 19-21 and to affinity membranes. 22The uses of lectins in contemporary glycoproteomics and glycomics are likely to grow due to the frequent needs for fractionation and preconcentration for high-sensitivity mass spectrometry (MS). 23 Although glycoproteins and glycopeptides could be fractionated through lectins with different specificity, in most reported cases, lectin affinity procedures were employed to isolate the entire pools of glycoconjugates rather than specific structures. 24,25 Consequently, wheat germ agglutinin and concanavalin A have been widely used because of their broad specificity. Although it is assumed that different glycoproteins produced by a certain cell type possess a similar type...

Hypoxia-induced Changes to Integrin α 3 Glycosylation Facilitate Invasion in Epidermoid Carcinoma Cell Line A431

“…The most commonly employed technique for the analysis of protein glycosylation is HPLC coupled to MS (42,43). A number of HPLC protocols have been developed that enable the enrichment of glycopeptides from complex samples, including lectin affinity (44,45), hydrazide reaction (46), ion exchange (47), hydrophilic-interaction chromatography (48), and TiO 2 (49). For quantitative analysis of glycoproteins, Zhou et al analyzed N-glycoprotein profiles in tear fluid using hydrazide-resin capture, iTRAQ labeling, and two-dimensional LC-MS/MS identification; a total of 43 unique N-glycoproteins were identified, and some were found to be modulated at several glycosites under disease conditions (50).…”

New hyphenated methodologies in high‐sensitivity glycoprotein analysis

“…Lectin unbound fractions (a, c, and e), and lectin bound fractions (b, d, and f). Reproduced from [100], with permission. Comparison of the number of unique and common proteins identified with/without serial lectin enrichment approach.…”

“…However, this procedure is not compatible with the online pressurized systems that are intended to minimize sample handling, contamination, and sample losses. To promote the analytical uses of immobilized lectins as a part of pressurized chromatographic systems, we have synthesized silica-based materials and evaluated their performance in glycoproteomic analysis [100]. More recently, the silica-based lectin microcolumns have been employed in proteomic studies on complex biological materials such as blood serum [100] and soluble extracts of rat brain [101].…”

“…A wide range of lectin materials have been successfully prepared including Con A, specific for mannose and glucose residues, Sambucus nigra (SNA), specific for α-(2-6)-sialylated structures, Phaseolus vulgaris (PHA-L), partially specific for carbohydrate containing Gal-β-(1-4)GlcNAc-β-(1,2) man sequence, and Ulex europaeus (UEA-I), specific for α-L-fucosylated moieties. Because of their high-pressure compatibility, these columns can readily be used in a valve-based analytical system [100].…”

Affinity Entrapment of Oligosaccharides and Glycopeptides Using Free Lectin Solution

“…10 More recently, the use of high-performance lectin-affinity enrichment of glycoprotein using a series of silica-bound lectins at microscale levels has been reported. 5,[11][12][13] This enrichment procedure was combined with nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS) to identify numerous glycoproteins. 13 A multi-lectin column prepared by mixing three lectin-immobilized gels was successively used in enrichment of serum glycoproteins.…”