Printed covalent glycan array for ligand profiling of diverse glycan binding proteins

Abstract

Here we describe a glycan microarray constructed by using standard robotic microarray printing technology to couple amine functionalized glycans to an amino-reactive glass slide. The array comprises 200 synthetic and natural glycan sequences representing major glycan structures of glycoproteins and glycolipids. The array has remarkable utility for profiling the specificity of a diverse range of glycan binding proteins, including C-type lectins, siglecs, galectins, anticarbohydrate antibodies, lectins from plants and microbes, and intact viruses.carbohydrate ͉ lectin ͉ microarray ͉ glycoprotein ͉ glycolipid

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

“…org). Version 3.1 of this array, consisting of 377 different glycans, was used to determine the specificity and relative affinity of PP2-A1-His 6 for oligosaccharides (Blixt et al, 2004). In this assay, protein binding was quantified with a fluorescent antibody against the His 6 tag present on the recombinant protein.…”

Posttranslational Protein Modifications

“…Although the recent developments and use of genomics and proteomics offer much promise in the search for molecular markers of early stage cancers, these methods are inadequate to probe the dynamic nature of signaling processes that cells exhibit during their transformation to become neoplastic. The diverse realm of posttranslational modification (PTM) 1 of proteins encompasses many of the critical signaling events occurring during neoplastic transformation. PTMs offer a plethora of candidates for biomarker detection that complement discoveries using strictly proteomics or genomics platforms.…”

“…Since that time some advancement has been made in technological developments to study PTMs, identifying the central roles they play in cancer progression, and determining their amenability as either a cancer biomarker or therapeutic target. A limiting factor in PTM research is that technologies to screen vast numbers of molecules for a particular type of modification are often not available, although recent developments of specific probes and multiplexed platforms may now make broader scale PTM surveying feasible (1)(2)(3)(4)(5)(6). For the most part, research in PTM as it relates to biomarker discovery has required the study of discreet modifications on specific proteins of importance to cancer biology.…”

The application of neoglycopeptides in the development of sensitive surface plasmon resonance-based biosensors

“…Although analytes can be detected by a wide range of immobilized ligands, such as antibodies (Soh et al, 2003;Liu et al, 2004;Choi et al, 2005;Muller-Renaud et al, 2005), enzymes, and carbohydrate-binding proteins (CBPs) (Hsieh et al, 2004), the development of carbohydrate-based biosensors has recently received increased attention because of the importance of carbohydrate-protein interactions in life. So far, carbohydratebased biosensors are predominantly used for the characterization of the kinetics and affinity of carbohydrate-protein interactions (Shinohara et al, 1994;Bourne et al, 2002;Zhang et al, 2006), or in an array-type set-up (Love and Seeberger, 2002;Feizi et al, 2003;Blixt et al, 2004;Dyukova et al, 2006) to determine the specificity, and possible cross-reactivity of a CBP. However, it would also be of interest to apply carbohydrate-based biosensors to the sensitive detection and quantification of minute amounts of CBPs, which could lead to the development of diagnostic tools.…”