Comparative Two-Dimensional Polyacrylamide Gel Electrophoresis (2D-PAGE) of Human Milk to Identify Dysregulated Proteins in Breast Cancer

Breast Cancer (BC) remains a major cause of mortality, and early detection is considered important for reducing BC-associated deaths. Early detection of BC is challenging in young women, due to the limitations of mammography on the dense breast tissue of young women. We recently reported results of a pilot study, using one-dimensional polyacrylamide gel electrophoresis (1D-PAGE)-based mass spectrometry (MS) to investigate differences in milk proteins from women with and without BC. Here, we applied two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)-based MS to compare the protein pattern in milk from the breasts of a single woman who was diagnosed with BC in one breast 24 months after donating her mil. Statistically different gel spots were picked for protein digestion followed by nanoLC-MS/MS analysis. The upregulated proteins in BC versus control are alpha-amylase, gelsolin isoform a precursor, alpha-2-glycoprotein 1 zinc isoform CRA_b partial, apoptosis-inducing factor 2 and vitronectin. Several proteins were downregulated in the milk of the breast later diagnosed with cancer as compared to the milk from the healthy breast, including different isoforms of albumin, cholesterol esterase, different isoforms of lactoferrin, different proteins from the casein family and different isoforms of lysozyme. Results warrant further studies to determine the usefulness of these milk proteins for assessing risk and detecting occult disease.

乳腺癌（Breast Cancer, BC）仍是全球致死率较高的恶性肿瘤之一，早期筛查被视作降低乳腺癌相关死亡的核心策略。年轻女性的乳腺癌早期筛查存在较大挑战，这是由于乳腺钼靶摄影对其致密型乳腺组织的检测效能有限。本团队此前曾报道过一项预实验成果：采用基于一维聚丙烯酰胺凝胶电泳（1D-PAGE）的质谱（MS）技术，对比了乳腺癌患者与健康女性的乳汁蛋白质组差异。本研究进一步应用基于二维聚丙烯酰胺凝胶电泳（2D-PAGE）的质谱技术，对一名女性的乳汁样本展开分析——该女性在捐献乳汁后的24个月，被确诊为单侧乳腺癌。研究人员对存在统计学显著性差异的凝胶斑点进行蛋白消化，随后通过纳流液相色谱-串联质谱（nanoLC-MS/MS）完成蛋白质鉴定。相较于健康侧乳腺的乳汁，后续确诊患癌侧乳腺的乳汁中存在多种上调蛋白，包括α-淀粉酶、凝溶胶蛋白亚型a前体、α-2-糖蛋白1锌亚型CRA_b部分序列、凋亡诱导因子2以及玻连蛋白；同时下调的蛋白则涵盖多种白蛋白亚型、胆固醇酯酶、多种乳铁蛋白亚型、多种酪蛋白家族蛋白以及多种溶菌酶亚型。本研究结果支持开展后续研究，以明确此类乳汁蛋白在乳腺癌风险评估与隐匿性疾病检测中的应用价值。