Neubauer Hans, Clare Susan E, Kurek Raffael, Fehm Tanja, Wallwiener Diethelm, Sotlar Karl, Nordheim Alfred, Wozny Wojciech, Schwall Gerhard P, Poznanović Slobodan, Sastri Chaturvedula, Hunzinger Christian, Stegmann Werner, Schrattenholz André, Cahill Michael A
University Women's Hospital, Tübingen, Germany.
Electrophoresis. 2006 May;27(9):1840-52. doi: 10.1002/elps.200500739.
The presence of progesterone receptor (PR) in estrogen receptor (ER)-positive breast cancer is associated with a good prognosis, and indicates that tumors are likely to respond to tamoxifen. However, ER+/PR- tumors respond less well. To reveal the potential molecular mechanism of this phenomenon, we sought to identify differential protein abundances between invasive ductal carcinoma cells from cryopreserved ER+/PR+ and ER+/PR- mammary tumor specimens. Because current proteomics methods are hampered in the examination of most primary human tumor samples by the extreme tissue heterogeneity, we used laser capture microdissection (LCM) to isolate tumor cells and developed a sample pooling strategy to analyze small sample protein lysates. Proteins from LCM-harvested tumors were pooled into four sub-pools from each condition of three tumors/sub-pool, and proteins from respective paired sub-pools were co-electrophoresed by 2-DE using 54-cm IEF over pH 4-9. Abundance ratios were accurately quantified by a differential multiplex radioactive ProteoTope method at low attomole levels ( approximately 3.6 microg protein per labeling reaction, <180 ng per multiplex protein sample per 54-cm gel). Applying this approach, differentially displayed proteins were identified by MS using comigrating non-radioactively labeled tumor proteins. They include decreased cytochrome b5 and transgelin, and more abundant CRABP-II, cyclophilin A, Neudesin, and hemoglobin in ER+/PR+ tumors versus ER+/PR- providing a possible explanation for differential susceptibility against tamoxifen as a result of deregulated cytochrome b5-dependent metabolism. This study demonstrates the potential of ProteoTope and LCM to enable extremely sensitive and precise differential analyses from well-defined primary clinical specimen.
雌激素受体（ER）阳性乳腺癌中孕激素受体（PR）的存在与良好预后相关，表明肿瘤可能对他莫昔芬有反应。然而，ER+/PR-肿瘤的反应较差。为了揭示这种现象的潜在分子机制，我们试图鉴定来自冷冻保存的ER+/PR+和ER+/PR-乳腺肿瘤标本的浸润性导管癌细胞之间的差异蛋白质丰度。由于当前的蛋白质组学方法在检查大多数原发性人类肿瘤样本时受到极端组织异质性的阻碍，我们使用激光捕获显微切割（LCM）分离肿瘤细胞，并开发了一种样本合并策略来分析小样本蛋白质裂解物。将LCM采集的肿瘤中的蛋白质从每种条件下的三个肿瘤/亚库中合并成四个亚库，并使用pH 4-9的54-cm等聚焦在2-DE上对来自各自配对亚库的蛋白质进行共电泳。通过差异多重放射性ProteoTope方法在低阿托摩尔水平（每个标记反应约3.6μg蛋白质，每54-cm凝胶的多重蛋白质样品<180 ng）准确量化丰度比。应用这种方法，使用共迁移的非放射性标记肿瘤蛋白通过质谱鉴定差异显示的蛋白质。与ER+/PR-肿瘤相比，ER+/PR+肿瘤中细胞色素b5和转胶蛋白减少，而CRABP-II、亲环蛋白A Neudesin和血红蛋白更丰富，这为细胞色素b5依赖性代谢失调导致对他莫昔芬的差异敏感性提供了可能的解释。本研究证明了ProteoTope和LCM在从明确的原发性临床标本中进行极其灵敏和精确的差异分析的潜力。
