Kulasingam Vathany, Diamandis Eleftherios P
Department of Laboratory Medicine and Pathobiology, Mount Sinai Hospital, University of Toronto, Toronto, Ontario M5G 1X5, Canada.
Mol Cell Proteomics. 2007 Nov;6(11):1997-2011. doi: 10.1074/mcp.M600465-MCP200. Epub 2007 Jul 25.
A "bottom-up" proteomics approach and a two-dimensional (strong cation exchange followed by reversed-phase) LC-MS/MS strategy on a linear ion trap (LTQ) were utilized to identify and compare expressions of extracellular and membrane-bound proteins in the conditioned media of three breast cell lines (MCF-10A, BT474, and MDA-MB-468). Proteomics analysis of the media identified in excess of 600, 500, and 700 proteins in MCF-10A, BT474, and MDA-MB-468, respectively. We successfully identified the internal control proteins, kallikreins 5, 6, and 10 (ranging in concentration from 2 to 50 microg/liter) in MDA-MB-468 conditioned medium as validated by ELISA and confidently identified Her-2/neu in BT474 cells. Subcellular localization was determined based on Genome Ontology terms for all the 1,139 proteins of which 34% were classified as extracellular and membrane-bound. Proteomics analysis of MDA-MB-468 cell lysate demonstrated that only 5% of all identified proteins were extracellular. This confirmed our hypothesis that examining the CM of cell lines, as opposed to the cell lysates, leads to a significant enrichment in secreted proteins. Tissue specificity, functional classifications, and spectral counting were performed. Elafin, a protease inhibitor, identified in the conditioned media of BT474 and MDA-MB-468 and the three kallikreins (KLK5, KLK6, and KLK10) were validated using an immunoassay on various serum and biological samples. Some of the secreted proteins identified have established roles in breast cancer development (cell growth, differentiation, and metastasis) and/or are linked to early onset breast cancer. Our approach to mining for low abundance molecules could identify proteins in various stages of breast cancer development. Many of the identified proteins are potentially useful to investigate as circulating serum breast cancer biomarkers.
采用“自下而上”的蛋白质组学方法和二维（强阳离子交换后反相）液相色谱-串联质谱策略，在线性离子阱（LTQ）上对三种乳腺癌细胞系（MCF-10A、BT474和MDA-MB-468）条件培养基中的细胞外蛋白和膜结合蛋白进行鉴定和比较。对培养基的蛋白质组学分析分别在MCF-10A、BT474和MDA-MB-468中鉴定出超过600、500和700种蛋白质。我们成功地在MDA-MB-468条件培养基中鉴定出内参蛋白激肽释放酶5、6和10（浓度范围为2至50微克/升），经酶联免疫吸附测定（ELISA）验证，并在BT474细胞中可靠地鉴定出Her-2/neu。基于基因本体术语确定了所有1139种蛋白质的亚细胞定位，其中34%被归类为细胞外蛋白和膜结合蛋白。对MDA-MB-468细胞裂解物的蛋白质组学分析表明，所有鉴定出的蛋白质中只有5%是细胞外的。这证实了我们的假设，即与细胞裂解物相比，检测细胞系的条件培养基会导致分泌蛋白的显著富集。进行了组织特异性、功能分类和光谱计数。在BT474和MDA-MB-468的条件培养基中鉴定出的蛋白酶抑制剂弹性蛋白酶和三种激肽释放酶（KLK5、KLK6和KLK10），通过对各种血清和生物样品进行免疫测定进行了验证。鉴定出的一些分泌蛋白在乳腺癌发展（细胞生长、分化和转移）中具有既定作用，和/或与早发性乳腺癌有关。我们挖掘低丰度分子的方法可以鉴定乳腺癌发展各个阶段的蛋白质。许多鉴定出的蛋白质可能作为循环血清乳腺癌生物标志物进行研究，具有潜在的应用价值。
