Glycan microarray for studying carbohydrate-protein interactions requires diverse classes of well-defined glycan standards. In this study, a purification strategy was established based on two-dimensional hydrophilic interaction liquid chromatography and porous graphitized carbon chromatography (HILIC × PGC) for the acquisition of neutral N-glycan standards from natural source. A total of thirty-one N-glycan compounds including seven pairs of isomers with the amounts from 0.7 to 230.0 nmol were isolated from ovalbumin as the model glycoconjugate. The purified N-glycans covered high-mannose, hybrid as well as multi-antenna asymmetric complex types. The purity of majority of these N-glycans was higher than 90%. Detailed structures of the N-glycan compounds were verified via negative ion tandem MS analysis, in which specific diagnostic ions including D- and E-ions were used to identify isomeric and terminal fine structures. The tag-free glycan compounds with well-defined structures, purity and amounts were finally assembled on the glass slide through neoglycolipid technology. Microarray binding assay of purified glycans with WGA lectin indicated the potential of the established strategy in glycan library expansion and functional glycomics.

用于研究碳水化合物与蛋白质相互作用的聚糖微阵列需要多种类别的定义明确的聚糖标准品。在这项研究中，建立了一种基于二维亲水相互作用液相色谱和多孔石墨化碳色谱（HILIC×PGC）的纯化策略，用于从天然来源中获取中性N-聚糖标准品。从卵清蛋白中分离出总共31种N-聚糖化合物，包括7对异构体，其量为0.7至230.0 nmol，作为模型糖缀合物。纯化的N-聚糖涵盖了高甘露糖，杂合以及多天线不对称复杂类型。这些N-聚糖的大多数纯度高于90％。通过负离子串联质谱分析验证了N-聚糖化合物的详细结构，其中包括D和E离子的特定诊断离子用于鉴定异构体和末端精细结构。最后，通过新糖脂技术将具有明确结构，纯度和数量的无标签聚糖化合物组装在载玻片上。纯化的聚糖与WGA凝集素的微阵列结合测定表明，建立聚糖库扩展和功能糖组学策略的潜力。