title: Nonreductive chemical release of intact N-glycans for subsequent labeling and analysis by mass spectrometry.
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.
authors.affiliation: Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi'an 710069, China.
authors.forename: Jiangbei
authors.initials: J
authors.lastname: Yuan
authors.affiliation: Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi'an 710069, China.
authors.forename: Chengjian
authors.initials: C
authors.lastname: Wang
authors.affiliation: Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi'an 710069, China.
authors.forename: Yujiao
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi'an 710069, China. Electronic address: huanglj@nwu.edu.cn.
authors.forename: Linjuan
authors.initials: L
authors.lastname: Huang
authors.affiliation: Educational Ministry Key Laboratory of Resource Biology and Biotechnology in Western China, College of Life Science, Northwest University, Xi'an 710069, China. Electronic address: wangzhf@nwu.edu.cn.
authors.forename: Zhongfu
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D053000:Analytic Sample Preparation Methods / Q000191:economics / Q000379:methods*; D000818:Animals; D000983:Antipyrine / Q000031:analogs & derivatives / Q000737:chemistry; D002417:Cattle; D003362:Cost-Benefit Analysis; D000077553:Edaravone; D005643:Fucose / Q000737:chemistry; D054794:Glycomics; D006023:Glycoproteins / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D013194:Staining and Labeling; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006023:Glycoproteins; D010940:Plant Proteins; D011134:Polysaccharides; D005643:Fucose; D000077553:Edaravone; D000983:Antipyrine
keywords: 1-Phenyl-3-methyl-5-pyrazolone; Electrospray ionization mass spectrometry; Glycoprotein; N-Glycan; Nonreductive release