Versatile fluorescent derivatization of glycans for glycomic analysis.
The new field of functional glycomics encompasses information about both glycan structure and recognition by carbohydrate-binding proteins (CBPs) and is now being explored through glycan array technology. Glycan array construction, however, is limited by the complexity of efficiently generating derivatives of free, reducing glycans with primary amines for conjugation. Here we describe a straightforward method to derivatize glycans with 2,6-diaminopyridine (DAP) to generate fluorescently labeled glycans (glycan-DAP conjugates or GDAPs) that contain a primary amine for further conjugation. We converted a wide variety of glycans, including milk sugars, N-glycans, glycosaminoglycans and chitin-derived glycans, to GDAPs, as verified by HPLC and mass spectrometry. We covalently conjugated GDAPs to N-hydroxysuccinimide (NHS)-activated glass slides, maleimide-activated protein, carboxylated microspheres and NHS-biotin to provide quantifiable fluorescent derivatives. All types of conjugated glycans were well-recognized by appropriate CBPs. Thus, GDAP derivatives provide versatile new tools for biologists to quantify and covalently capture minute quantities of glycans for exploring their structures and functions and generating new glycan arrays from naturally occurring glycans.
Xia
Baoyun
B
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, 975 N.E. 10th St., BRC417, Oklahoma City, Oklahoma 73104, USA.
Kawar
Ziad S
ZS
Ju
Tongzhong
T
Alvarez
Richard A
RA
Sachdev
Goverdhan P
GP
Cummings
Richard D
RD
D019921:Biotinylation; D002236:Carbohydrate Conformation; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002686:Chitin / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D005453:Fluorescence; D013058:Mass Spectrometry; D046228:Microarray Analysis / Q000295:instrumentation* / Q000379:methods*; D008863:Microspheres; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D011725:Pyridines / Q000737:chemistry
D002352:Carrier Proteins; D011134:Polysaccharides; D011725:Pyridines; D002686:Chitin; C505797:2,6-diaminopyridine