Glycan Array on Aluminum Oxide-Coated Glass Slides through Phosphonate Chemistry

Abstract

A new type of glycan array covalently or noncovalently attached to aluminum oxide-coated glass (ACG) slides has been developed for studies of enzymatic reactions and protein binding. To prepare the noncovalent array, glycans with a polyfluorinated hydrocarbon (-C(8)F(17)) tail are spotted robotically onto the ACG slide surface containing a layer of polyfluorinated hydrocarbon terminated with phosphonate. After incubation and washing, the noncovalent array can be characterized by MS-TOF via ionization/desorption at a low laser energy without addition of matrix. A representative cellotetraose array was developed to study the activity and specificity of different cellulases and to differentiate the exo- and endoglucanase activities. To prepare the covalent array, glycans with a phosphonic acid tail were synthesized and spotted robotically onto the ACG slide surface. After incubation, the slides can be used directly for quantitative protein binding analysis. Compared to the preparation of glycan arrays on glass slides and other surfaces, this method of arraying using phosphonic acid reacting with ACG is more direct, convenient, and effective and represents a new platform for the high-throughput analysis of protein-glycan interactions.

Therapeutic Antibodies to Ganglioside GD2 Evolved from Highly Selective Germline Antibodies

“…Of particular relevance to this study, many of the array components contain glycan sequences that are closely related to GD2 such as GD1a, GD1b, GD3, GM1a, GM1b, GM2, GM3, GT1a, GT2, GT3, GQ2, and GA2 (Figure 1). We (Gildersleeve et al, 2008; Gildersleeve and Wright, 2016; Oyelaran et al, 2009; Shoreibah et al, 2011; Zhang et al, 2010) and others (Chang et al, 2010; Dupin et al, 2015; Godula and Bertozzi, 2012; Goudot et al, 2013; Hung et al, 2013; Jaipuri et al, 2008; Karamanska et al, 2008; Ligeour et al, 2015; Park and Shin, 2007; Shivatare et al, 2013; Song et al, 2008; Wang et al, 2008) have used glycan microarrays to evaluate apparent K D values for carbohydrate-protein interactions and have found good agreement with previously published values measured by surface plasmon resonance.…”

Discovery of glycosyltransferases using carbohydrate arrays and mass spectrometry

“…A related approach was used to assess the specificity of a polypeptide GalNAc transferase using an array of peptides immobilized on SAMs 27 . Other work used MALDI-TOF mass spectrometry to identify reaction products of glycan arrays on an aluminum oxide-coated glass slide 28 and of alkyl-glycans that were immobilized onto monolayers using hydrophobic interactions 29 . The example we describe in the present work is significant because it integrates laboratory automation protocols with mass spectrometry to efficiently perform tens of thousands of reactions for the purpose of discovering novel GTs.…”

Microarray-Based Technology for Glycomics Analysis

“…Recently, Wong and coworkers developed novel noncovalent carbohydrate microarray on aluminum oxide-coated glass (ACG) slides. In this case, sugars with a polyfluorinated hydrocarbon (-C 8 F 17 ) tail are spotted robotically onto the highly hydrophobic ACG slide surface containing a layer of polyfluorinated hydrocarbon terminated with phosphonate [35]. However, the noncovalent binding is not strong enough to yield stable microarrays capable of standing the necessary washing steps and incubation conditions in subsequent reactions.…”