Ratiometric Lectin Microarray Analysis of the Mammalian Cell Surface Glycome

Abstract

The mammalian cell surface is rich with carbohydrate polymers involved in a diversity of biological recognition events. Dynamic alterations of surface glycans mediate cell-cell communication in the immune system and host specificity of bacterial and viral pathogens. In addition, altered surface glycosylation has been implicated in disease progression of many cancers and may serve as important new targets for therapeutics. Despite the importance of glycosylation, the systematic analysis of sugars, i.e., glycomics, has lagged behind the well-studied disciplines of genomics and proteomics. This deficiency is due in part to the unique analytical challenges presented by glycans and the overwhelming diversity of sugars in nature. New microarray technologies have provided a high-throughput methods with which to probe the glycome. Our laboratory has pioneered a shown ratiometric two-color lectin microarray method that rapidly evaluates differences in the glycosylation of mammalian cells. Herein, we present a detailed protocol of our lectin microarray methodology for the differential analysis of mammalian glycomes.

miRNA proxy approach reveals hidden functions of glycosylation

“…Cell sample preparation and lectin microarray analysis then were conducted as previously described (45). Briefly, cell membrane samples were prepared and labeled with NHS-Cy5.…”

A Systems Biology Approach Identifies FUT8 as a Driver of Melanoma Metastasis

“…Samples were deparaffinized with xylene and ethanol, rehydrated and solubilized in Cy buffer (0.1 M NaHCO 3 , pH=9.3) containing 0.5% NP40 (Nonidet P-40) and labeled as previously described (Hsu et al, 2011; Pilobello et al, 2013). Reference sample was a mixture of 6 primary and 6 metastatic samples.…”

“…Total protein concentration was quantified by BCA assay. Samples were then labeled with NHS-Cy3 or -Cy5 and analyzed on our lectin microarrays in dual color using standard protocols (Hsu et al, 2011; Pilobello et al, 2013). Reference sample was a mixture of 6 primary and 6 metastatic samples.…”

Comparison of orthogonal chromatographic and lectin-affinity microarray methods for glycan profiling of a therapeutic monoclonal antibody

“…The glycoprofiling lectin microarray is an innovative medium-to highthroughput format reported almost simultaneously by different groups [65,70,107,108]. The method employs slides spotted with lectin proteins which selectively bind to specific carbohydrate structures, particularly their non-reducing termini, based on their constituent monosaccharides and the configuration of the glycosidic linkages [67][68][69][71][72][73]76,77]. Microarrays for particular glycomics or glycoproteomics applications, to evaluate and/or quantitate specific glycosylation patterns, may thus be devised by the judicious selection of lectins and optimization of lectin panels.…”

“…However, both HPAEC-PAD and HILIC-FD require removal of glycans from the underlying protein prior to analysis, involve multiple steps are overall rather time-consuming and relatively expensive, demand significant operator experience and expertise, and involve considerable data analysis. In contrast, lectin microarrays provide medium-throughput glycan fingerprinting information using comparatively inexpensive scanner platforms already available in many laboratories [65][66][67][68][69][70][71][72][73][74][75][76][77]. Proficient operation is achieved with significantly less training and experience than other methods.…”