Lee Ling Y, Thaysen-Andersen Morten, Baker Mark S, Packer Nicolle H, Hancock William S, Fanayan Susan
Department of Chemistry and Biomolecular Sciences and ‡Australian School of Advanced Medicine, Macquarie University , Sydney, NSW 2109, Australia.
J Proteome Res. 2014 Nov 7;13(11):4783-95. doi: 10.1021/pr500331m. Epub 2014 Sep 11.
The secreted cellular sub-proteome (secretome) is a rich source of biologically active glycoproteins. N-Glycan profiling of secretomes of cultured cancer cells provides an opportunity to investigate the link between protein N-glycosylation and tumorigenesis. Utilizing carbon-LC-ESI-CID-MS/MS of protein released native N-glycans, we accurately profiled the secretome N-glycosylation of six human epithelial breast cells including normal mammary epithelial cells (HMEC) and breast cancer cells belonging to luminal A subtype (MCF7), HER2-overexpressing subtype (SKBR3), and basal B subtype (MDA-MB157, MDA-MB231, HS578T). On the basis of intact molecular mass, LC retention time, and MS/MS fragmentation, a total of 74 N-glycans were confidently identified and quantified. The secretomes comprised significant levels of highly sialylated and fucosylated complex type N-glycans, which were elevated in all cancer cells relative to HMEC (57.7-87.2% vs 24.9%, p < 0.0001 and 57.1-78.0% vs 38.4%, p < 0.0001-0.001, respectively). Similarly, other glycan features were found to be altered in breast cancer secretomes including paucimannose and complex type N-glycans containing bisecting β1,4-GlcNAc and LacdiNAc determinants. Subtype-specific glycosylation were observed, including the preferential expression of α2,3-sialylation in the basal B breast cancer cells. Pathway analysis indicated that the regulated N-glycans were biosynthetically related. Tight clustering of the breast cancer subtypes based on N-glycome signatures supported the involvement of N-glycosylation in cancer. In conclusion, we are the first to report on the secretome N-glycosylation of a panel of breast epithelial cell lines representing different subtypes. Complementing proteome and lipid profiling, N-glycome mapping yields important pieces of structural information to help understand the biomolecular deregulation in breast cancer development and progression, knowledge that may facilitate the discovery of candidate cancer markers and potential drug targets.
分泌型细胞亚蛋白质组（分泌蛋白质组）是生物活性糖蛋白的丰富来源。对培养的癌细胞分泌蛋白质组进行N-聚糖分析，为研究蛋白质N-糖基化与肿瘤发生之间的联系提供了契机。利用蛋白质释放的天然N-聚糖的碳-液相色谱-电喷雾电离-碰撞诱导解离串联质谱（carbon-LC-ESI-CID-MS/MS），我们准确分析了六种人乳腺上皮细胞的分泌蛋白质组N-糖基化情况，包括正常乳腺上皮细胞（HMEC）以及属于腔面A型亚型（MCF7）、HER2过表达亚型（SKBR3）和基底B型亚型（MDA-MB157、MDA-MB231、HS578T）的乳腺癌细胞。基于完整分子量、液相色谱保留时间和串联质谱碎片，共可靠鉴定和定量了74种N-聚糖。分泌蛋白质组包含大量高度唾液酸化和岩藻糖基化的复合型N-聚糖，相对于HMEC，所有癌细胞中这些聚糖均有所升高（分别为57.7 - 87.2%对24.9%，p < 0.0001；57.1 - 78.0%对38.4%，p < 0.0001 - 0.001）。同样，在乳腺癌分泌蛋白质组中还发现其他聚糖特征发生了改变，包括寡甘露糖以及含有平分型β1,4- GlcNAc和LacdiNAc决定簇的复合型N-聚糖。观察到了亚型特异性糖基化，包括基底B型乳腺癌细胞中α2,3-唾液酸化的优先表达。通路分析表明，受调控的N-聚糖在生物合成上相关。基于N-糖组特征对乳腺癌亚型进行的紧密聚类支持了N-糖基化与癌症的关联。总之，我们首次报道了一组代表不同亚型的乳腺上皮细胞系的分泌蛋白质组N-糖基化情况。与蛋白质组和脂质分析相辅相成，N-糖组图谱提供了重要的结构信息，有助于理解乳腺癌发生和发展过程中的生物分子失调，这些知识可能有助于发现候选癌症标志物和潜在药物靶点。
