A relatively novel activation technique, electron-transfer/higher-energy collision dissociation (EThcD) was used in the LC-MS/MS analysis of tryptic glycopeptides enriched with wheat germ agglutinin from human urine samples. We focused on the characterization of mucin-type O-glycopeptides. EThcD in a single spectrum provided information on both the peptide modified and the glycan carried. Unexpectedly, glycan oxonium ions indicated the presence of O-acetyl, and even O-diacetyl-sialic acids. B and Y fragment ions revealed that (i) in core 1 structures the Gal residue featured the O-acetyl-sialic acid, when there was only one in the glycan; (ii) several glycopeptides featured core 1 glycans with disialic acids, in certain instances O-acetylated; (iii) the disialic acid was linked to the GalNAc residue whatever the degree of O-acetylation; (iv) core 2 isomers with a single O-acetyl-sialic acid were chromatographically resolved. Glycan fragmentation also helped to decipher additional core 2 oligosaccharides: a LacdiNAc-like structure, glycans carrying sialyl Lewis^X/A at different stages of O-acetylation, and blood antigens. A sialo core 3 structure was also identified. We believe this is the first study when such structures were characterized from a very complex mixture and were linked not only to a specific protein, but also the sites of modifications have been determined.

中文翻译：

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电子尿素/高能碰撞解离法发现并表征了人类尿糖蛋白的唾液酸化O-糖基扩展库

LC-MS / MS分析中使用了一种相对较新的活化技术，即电子转移/高能碰撞解离（EThcD），富含人胚尿样中的小麦胚芽凝集素。我们专注于粘蛋白型O-糖肽的表征。单一光谱中的EThcD可提供有关修饰的肽和携带的聚糖的信息。出乎意料的是，聚糖氧鎓离子表明存在O-乙酰基，甚至O-二乙酰基唾液酸。B和Y碎片离子表明：（i）在核心1的结构中，当聚糖中只有一个时，Gal残基具有O-乙酰基唾液酸；（ii）在某些情况下，一些糖肽的核心1聚糖带有二唾液酸，在某些情况下是O-乙酰化的；（iii）无论O-乙酰化程度如何，二唾液酸都与GalNAc残基连接；（iv）色谱分离具有单个O-乙酰基唾液酸的核心2异构体。聚糖片段化还有助于破译额外的核心2低聚糖：LacdiNAc样结构，带有唾液酸路易斯的聚糖在O-乙酰化和血液抗原不同阶段的^{X / A。}还确定了唾液酸核3的结构。我们相信这是首次研究，当这种结构由非常复杂的混合物表征并且不仅与特定蛋白质相关，而且修饰位点已经确定时。