Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection
Breast cancer proteomics by laser capture microdissection, sample pooling, 54-cm IPG IEF, and differential iodine radioisotope detection
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DOI：
10.1002/elps.200500739
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发表时间：
2006-05-01
期刊：
影响因子：
2.9
通讯作者：
Cahill, Michael A.
中科院分区：
文献类型：
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作者：
Neubauer, Hans;Clare, Susan E.;Cahill, Michael A.
雌激素受体（ER）阳性乳腺癌中孕激素受体（PR）的存在与良好的预后相关，并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，我们试图鉴定冷冻保存的 ER+/PR+ 和 ER+/PR- 乳腺肿瘤标本中侵袭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）来分离肿瘤细胞，并开发了样本池策略来分析小样本蛋白质裂解物。将来自 LCM 收获的肿瘤的蛋白质汇集到来自三个肿瘤/子池的每种条件的四个子池中，并且使用 54-cm IEF 在 pH 4-9 上通过 2-DE 对来自各自配对子池的蛋白质进行共电泳。通过微分多重放射性 ProteoTope 方法在低阿托摩尔水平下准确量化丰度比（“类似于每个标记反应 3.6 μg 蛋白质，每 54 厘米凝胶每个多重蛋白质样品 < 180 ng）”。应用这种方法，通过 MS 使用共迁移的非放射性标记的肿瘤蛋白来鉴定差异展示的蛋白。它们包括与 ER+/PR(-) 相比，ER+/PR+ 肿瘤中细胞色素 b(5) 和转凝胶蛋白减少，以及更丰富的 CRABP-II、亲环蛋白 A、Neudesin 和血红蛋白，这为细胞色素 b(5) 依赖性代谢失调导致对他莫昔芬的敏感性差异提供了可能的解释。这项研究证明了 ProteoTope 和 LCM 能够对明确的主要临床样本进行极其灵敏和精确的差异分析。
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 subpools 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 (`similar to 3.6 mu g 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 b(5) 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 b(5)-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.