Mucin-type O-glycans play key roles in many cellular processes, and they are often altered in human diseases. A major challenge in studying the role of O-glycans through functional O-glycomics is the absence of a complete repertoire of the glycans that comprise the human O-glycome. Here we describe a cellular O-glycome preparation strategy, Preparative Cellular O-Glycome Reporter/Amplification (pCORA), that introduces 4-N₃-Bn-GalNAc(Ac)₃ as a novel precursor in large-scale cell cultures to generate usable amounts of O-glycans as a potential O-glycome factory. Cultured human non-small cell lung cancer (NSCLC) A549 cells take up the precursor, which is extended by cellular glycosyltransferases to produce 4-N₃-Bn-α-O-glycans that are secreted into the culture medium. The O-glycan derivatives can be clicked with a fluorescent bifunctional tag that allows multidimensional HPLC purification and production of a tagged glycan library, representing the O-glycome of the corresponding cells. We obtained ∼5% conversion of precursor to O-glycans and purified a tagged O-glycan library of over 100 O-glycan derivatives, many of which were present in >100 nmol amounts and were sequenced by sequential MS fragmentation (MSⁿ). These O-glycans were successfully printed onto epoxy glass slides as an O-glycome shotgun microarray. We used this novel array to explore binding activity of serum IgM in healthy persons and NSCLC patients at different cancer stages. This novel strategy provides access to complex O-glycans in significant quantities and may offer a new route to discovery of potential diagnostic disease biomarkers.

中文翻译：

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功能性糖蛋白的细胞O-糖蛋白的扩增和制备。

粘蛋白型O聚糖在许多细胞过程中起着关键作用，并且在人类疾病中经常发生改变。通过功能性O-糖组学研究O-聚糖的作用的主要挑战是缺乏构成人O-糖组的聚糖的完整库。在这里，我们描述了一种细胞O型糖制备策略，即制备型细胞O型糖报告基因/扩增（pCORA），它在大规模细胞培养中引入4-N ₃ -Bn-GalNAc（Ac）₃作为新型前体以产生有用的潜在的O-糖工厂的数量。培养的人类非小细胞肺癌（NSCLC）A549细胞吸收前体，该前体通过细胞糖基转移酶扩展产生4-N ₃分泌到培养基中的-Bn-α-O-聚糖。可以用荧光双功能标签点击O-聚糖衍生物，该标签可以进行多维HPLC纯化并生成标记的聚糖文库，代表相应细胞的O-糖基。我们获得了约5％的前体转化为O-聚糖的纯度，并纯化了超过100种O-聚糖衍生物的标记O-聚糖文库，其中许多以> 100 nmol的量存在，并通过顺序MS片段化进行测序（MS ⁿ）。这些O-聚糖成功地作为O-糖基shot弹枪微阵列印刷在环氧玻璃载玻片上。我们使用这种新颖的阵列来探索健康人和处于不同癌症阶段的NSCLC患者血清IgM的结合活性。这种新颖的策略提供了获取大量复杂O-聚糖的途径，并可能为发现潜在诊断性疾病生物标记物提供一条新途径。