Generation of a natural glycan microarray using 9-fluorenylmethyl chloroformate (FmocCl) as a cleavable fluorescent tag.
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 alpha1,3-fucose and core alpha1,2-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.
Song
Xuezheng
X
Department of Biochemistry, Emory University School of Medicine, O. Wayne Rollins Research Center, 1510 Clifton Road, Suite 4001, Atlanta, GA 30322, USA.
Lasanajak
Yi
Y
Rivera-Marrero
Carlos
C
Luyai
Anthony
A
Willard
Margaret
M
Smith
David F
DF
Cummings
Richard D
RD
D000818:Animals; D005449:Fluorenes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry*; D006020:Glycopeptides / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D006023:Glycoproteins / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000378:metabolism; D007074:Immunoglobulin G / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D007075:Immunoglobulin M / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents; D051379:Mice; D008810:Mice, Inbred C57BL; D046228:Microarray Analysis / Q000379:methods*; D011134:Polysaccharides / Q000032:analysis*; D011402:Pronase / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005449:Fluorenes; D005456:Fluorescent Dyes; D006020:Glycopeptides; D006023:Glycoproteins; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007202:Indicators and Reagents; D011134:Polysaccharides; C054007:1-(9-fluorenyl)methyl chloroformate; D006735:Horseradish Peroxidase; D011402:Pronase
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