Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, 513 Parnassus Avenue, Box 0665, San Francisco, California 94143, United States.
J Proteome Res. 2012 Apr 6;11(4):2508-20. doi: 10.1021/pr201206w. Epub 2012 Mar 13.
We used a lectin chromatography/MS-based approach to screen conditioned medium from a panel of luminal (less aggressive) and triple negative (more aggressive) breast cancer cell lines (n=5/subtype). The samples were fractionated using the lectins Aleuria aurantia (AAL) and Sambucus nigra agglutinin (SNA), which recognize fucose and sialic acid, respectively. The bound fractions were enzymatically N-deglycosylated and analyzed by LC-MS/MS. In total, we identified 533 glycoproteins, ∼90% of which were components of the cell surface or extracellular matrix. We observed 1011 glycosites, 100 of which were solely detected in ≥3 triple negative lines. Statistical analyses suggested that a number of these glycosites were triple negative-specific and thus potential biomarkers for this tumor subtype. An analysis of RNaseq data revealed that approximately half of the mRNAs encoding the protein scaffolds that carried potential biomarker glycosites were up-regulated in triple negative vs luminal cell lines, and that a number of genes encoding fucosyl- or sialyltransferases were differentially expressed between the two subtypes, suggesting that alterations in glycosylation may also drive candidate identification. Notably, the glycoproteins from which these putative biomarker candidates were derived are involved in cancer-related processes. Thus, they may represent novel therapeutic targets for this aggressive tumor subtype.
我们采用凝集素色谱/MS 方法，筛选来自腔面（侵袭性较低）和三阴性（侵袭性较高）乳腺癌细胞系的条件培养基（n=5/亚型）。使用识别岩藻糖和唾液酸的凝集素 Aleuria aurantia (AAL) 和 Sambucus nigra agglutinin (SNA) 将样本进行分段。结合的部分经酶促 N-去糖基化后，通过 LC-MS/MS 进行分析。总共鉴定出 533 种糖蛋白，其中约 90%是细胞表面或细胞外基质的组成部分。我们观察到 1011 个糖基化位点，其中 100 个仅在≥3 个三阴性系中检测到。统计分析表明，其中一些糖基化位点是三阴性特异性的，因此可能是这种肿瘤亚型的潜在生物标志物。对 RNaseq 数据的分析表明，携带潜在生物标志物糖基化位点的蛋白质支架的编码 mRNA 中约有一半在三阴性与腔面细胞系中上调，并且编码岩藻糖基或唾液酸转移酶的一些基因在两种亚型之间表达差异，这表明糖基化的改变也可能驱动候选物的鉴定。值得注意的是，这些候选潜在生物标志物所来源于的糖蛋白参与癌症相关过程。因此，它们可能代表这种侵袭性肿瘤亚型的新治疗靶点。
