Lectin Chromatography/Mass Spectrometry Discovery Workflow Identifies Putative Biomarkers of Aggressive Breast Cancers

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.

本研究采用基于凝集素色谱-质谱（lectin chromatography/MS）的方法，对一组腔面型（侵袭性较弱）与三阴性（侵袭性较强）乳腺癌细胞系（每个亚型含5株细胞系）的条件培养基开展筛选。
实验使用识别岩藻糖的橙黄网孢盘菌凝集素（Aleuria aurantia, AAL）以及识别唾液酸的接骨木凝集素（Sambucus nigra agglutinin, SNA）对样本进行分级分离。结合组分经酶法N-去糖基化修饰后，采用液相色谱-串联质谱（LC–MS/MS）完成分析。
本研究共鉴定出533种糖蛋白，其中约90%为细胞表面或细胞外基质组分。
本研究共检测到1011个糖基化位点（glycosites），其中100个仅在至少3株三阴性乳腺癌细胞系中被检出。
统计学分析显示，上述糖基化位点中有多个为三阴性乳腺癌特异性位点，可作为该肿瘤亚型的潜在生物标志物。
对RNA测序（RNaseq）数据的分析显示，编码携带潜在生物标志物糖基化位点的蛋白骨架的mRNA中，约有一半在三阴性乳腺癌细胞系中较腔面型细胞系出现上调；同时多个编码岩藻糖基转移酶或唾液酸转移酶的基因在两种亚型间存在差异表达，提示糖基化修饰改变或可推动候选生物标志物的鉴定过程。
值得注意的是，这些推定的候选生物标志物来源糖蛋白均参与癌症相关生物学过程，因此它们或可成为该侵袭性肿瘤亚型的新型治疗靶点。