Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry |
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Authors: | Jiangbei Yuan Chengjian WangYujiao Sun Linjuan Huang Zhongfu Wang |
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Affiliation: | Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi’an 710069, China |
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Abstract: | A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate–peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI–MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies. |
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Keywords: | Glycoprotein Nonreductive release N-Glycan 1-Phenyl-3-methyl-5-pyrazolone Electrospray ionization mass spectrometry |
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