Reductive chemical release of N-glycans as 1-amino-alditols and subsequent 9-fluorenylmethyloxycarbonyl labeling for MS and LC/MS analysis.
Glycoproteins play pivotal roles in a series of biological processes and their glycosylation patterns need to be structurally and functionally characterized. However, the lack of versatile methods to release N-glycans as functionalized forms has been undermining glycomics studies. Here a novel method is developed for dissociation of N-linked glycans from glycoproteins for analysis by MS and online LC/MS. This new method employs aqueous ammonia solution containing NaBH3CN as the reaction medium to release glycans from glycoproteins as 1-amino-alditol forms. The released glycans are conveniently labeled with 9-fluorenylmethyloxycarbonyl (Fmoc) and analyzed by ESI-MS and online LC/MS. Using the method, the neutral and acidic N-glycans were successfully released without peeling degradation of the core α-1,3-fucosylated structure or detectable de-N-acetylation, revealing its general applicability to various types of N-glycans. The Fmoc-derivatized N-glycans derived from chicken ovalbumin, Fagopyrum esculentum Moench Pollen and FBS were successfully analyzed by online LC/MS to distinguish isomers. The 1-amino-alditols were also permethylated to form quaternary ammonium cations at the reducing end, which enhance the MS sensitivity and are compatible with sequential multi-stage mass spectrometry (MSn) fragmentation for glycan sequencing. The Fmoc-labeled N-glycans were further permethylated to produce methylated carbamates for determination of branches and linkages by sequential MSn fragmentation.


SIGNIFICANCE OF THE STUDY
N-Glycosylation represents one of the most common post-translational modification forms and plays pivotal roles in the structural and functional regulation of proteins in various biological activities, relating closely to human health and diseases. As a type of informational molecule, the N-glycans of glycoproteins participate directly in the molecular interactions between glycan epitopes and their corresponding protein receptors. Detailed structural and functional characterization of different types of N-glycans is essential for understanding the functional mechanisms of many biological activities and the pathologies of many diseases. Here we describe a simple, versatile method to indistinguishably release all types of N-glycans as functionalized forms without remarkable side reactions, enabling convenient, rapid analysis and preparation of released N-glycans from various complex biological samples. It is very valuable for studies on the complicated structure-function relationship of N-glycans, as well as for the search of N-glycan biomarkers of some major diseases and N-glycan related targets of some drugs.
['Wang|Chengjian|C|', 'Qiang|Shan|S|', 'Jin|Wanjun|W|', 'Song|Xuezheng|X|', 'Zhang|Ying|Y|', 'Huang|Linjuan|L|', 'Wang|Zhongfu|Z|']
D000818:Animals D002384:Catalysis D002645:Chickens D002853:Chromatography, Liquid D019612:Fagopyrum D005449:Fluorenes D054794:Glycomics D006023:Glycoproteins D013058:Mass Spectrometry D010047:Ovalbumin D010084:Oxidation-Reduction D011058:Pollen D011134:Polysaccharides D021241:Spectrometry, Mass, Electrospray Ionization D013194:Staining and Labeling D013402:Sugar Alcohols D053719:Tandem Mass Spectrometry
Reductive chemical release N-glycans LC/MS Fmoc Permethylation