Generation of a Natural Glycan Microarray Using 9-Fluorenylmethyl Chloroformate (FmocCl) as a Cleavable Fluorescent Tag

• Type of Material: Article
• Authors: Xuezheng Song, Emory University; Yi Lasanajak, Emory University; Carlos Rivera-Marrero, Emory University; Anthony Luyai, Emory University; Margaret Willard, Emory University; David Smith, Emory University; Richard Cummings, Emory University
• Language: English
• Date: 2009-12-15
• Publisher: Elsevier Masson
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2009, Elsevier
• License: Creative Commons Attribution-NonCommerical-NoDerivs 3.0 Unported
• Final Published Version (URL): http://dx.doi.org/10.1016/j.ab.2009.08.024
• Title of Journal or Parent Work: Analytical Biochemistry
• ISSN: 0003-2697
• Volume: 395
• Issue: 2
• Start Page: 151
• End Page: 160
• Grant/Funding Information: This work was supported by in part by a Bridge Grant to R.D.C from the Consortium for Functional Glycomics under NIGMS through NIH Grant GM62116 and by NIH Grant RO1AI47214 to R.D.C.
• Keywords: functional glycomics; Glycan array; immobilization; fluorescent labeling
• Research Categories: Chemistry, Biochemistry; Health Sciences, General

Abstract

Glycan microarray technology has become a successful tool for studying protein-carbohydrate interactions, but a limitation has been the laborious synthesis of glycan structures by enzymatic and chemical methods. Here we describe a new method to generate quantifiable glycan libraries from natural sources by combining widely used protease digestion of glycoproteins and Fmoc chemistry. Glycoproteins including chicken ovalbumin, bovine fetuin, and horseradish peroxidase (HRP) were digested by pronase, protected by FmocCl, and efficiently separated by 2D-HPLC. We show that glycans from HRP glycopeptides separated by HPLC and fluorescence monitoring retained their natural reducing end structures, mostly core α1,3-fucose and core α1,2-xylose. After simple Fmoc-deprotection, the glycans were printed on NHS-activated glass slides. The glycans were interrogated using plant lectins and antibodies in sera from mice infected with Schistosoma mansoni, which revealed the presence of both IgM and IgG antibody responses to HRP-glycopeptides. This simple approach to glycopeptide purification and conjugation allows for the development of natural glycopeptide microarrays without the need to remove and derivatize glycans and potentially compromise their reducing end determinants.