化学
聚糖
糖复合物
次氯酸
糖基化
糖肽
硫酸化
生物化学
氧化磷酸化
质谱法
苏氨酸
乙酰化
组合化学
丝氨酸
色谱法
糖蛋白
磷酸化
基因
抗生素
作者
Gaël M. Vos,Júlia Wéber,Igor R. Sweet,Kevin C. Hooijschuur,Javier Sastre Toraño,Geert‐Jan Boons
标识
DOI:10.1021/acs.analchem.3c00127
摘要
Protein O-glycosylation is one of the most diverse post-translational modifications. A critical step in the analysis of O-glycomes is the release of glycans from glycoconjugates. Current release methods rely mainly on β-elimination, which can result in peeling reactions and loss of base-sensitive functionalities leading to misidentification of glycans. To address this challenge, well-defined synthetic glycopeptides were used to establish a robust workflow for the oxidative release of O-glycans suitable for glycomics. Treatment of O-glycopeptides with neutralized hypochlorite resulted in the selective formation of lactic/glycolic acid glycosides, thereby retaining unique information of the parent amino acid (serine/threonine) that is lost by β-elimination. It locks the glycan in a closed ring configuration, thereby preventing peeling, and furthermore, the carboxylate of the anomeric tag promotes ionization in negative ion mode mass spectrometry, thereby increasing signal intensities. Labile modifications such as sialic acids, sulfates, and acetyl esters are maintained during the release procedure. The promise of the approach was demonstrated by the analysis of O-glycans from bovine submaxillary mucin, which identified mono- and di-O-acetylated sialoglycans as well as previously undetected tri-O-acetylated and sulfated glycans. The use of well-defined glycopeptide standards made it also possible to identify reaction intermediates, which in turn allowed us to postulate a reaction mechanism for oxidative O-glycan release under neutral conditions.
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