Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling
Proteomic analysis of ductal carcinoma of the breast using laser capture microdissection, LC-MS, and 16O/18O isotopic labeling
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DOI：
10.1021/pr034131l
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发表时间：
2004-05-01
影响因子：
4.4
通讯作者：
Karger, BL
中科院分区：
文献类型：
--
作者：
Zang, L;Toy, DP;Karger, BL
本研究的目标是开发一种定量表达蛋白质组学的方法，该方法通过激光捕获显微解剖（LCM）获得有限的样品量，例如，类似于10,000个细胞，通常含有大约1-4个杯子的蛋白质。选择O-15/O-18标记法作为测量差异表达的方法。首先为LCM细胞样品制定了包括裂解、消化和O-16/O-18标记在内的样品制备方案。选择的方案使用两个LCM帽检查10000个浸润性乳腺导管癌细胞，并证明是可重复的。进一步采用LC-IT-MS/MS结合O-16/O-18消化后标记法对低水平样品进行检测，首先对单个蛋白（BSA）进行检测，然后对5种不同蛋白量的蛋白质混合物进行检测，每种蛋白质的总含量约为1杯。接下来，使用所开发的方法比较微解剖正常导管上皮和转移性导管癌各10000个细胞的蛋白表达。从微解剖细胞中提取蛋白质，沉淀，用胰蛋白酶消化，然后标记O-16/O-18。正常细胞和转移细胞样品采用离子阱质谱反相LC-ESI-MS/MS进行分析。共鉴定出76个蛋白。线粒体异柠檬酸清氢酶、肌动蛋白和14-3-3蛋白zeta/delta在乳腺肿瘤细胞中显著上调。
The goal of this study was the development of a method for quantitative expression proteomics on the limited sample amounts obtained through laser capture microdissection (LCM) of tissues, e.g., similar to10 000 cells, which typically contain roughly 1-4 mug protein. The O-15/O-18 labeling method was selected as an approach to measure differential expression. A sample preparation protocol including lysis, digestion and O-16/O-18 labeling was first developed for LCM cell samples. The selected protocol was examined using two LCM caps of 10 000 cells from invasive ductal carcinoma of the breast and shown to be repeatable. A further test of LC-IT-MS/MS in combination with the O-16/O-18 post-digestion labeling method for studying low level samples was conducted first on a single protein (BSA) and then on a 5-standard protein mixture digest of different protein amounts, each with a total content approximately 1 mug. Next, protein expression was compared between 10 000 Cells, each of microdissected normal ductal epithelium and metastatic ductal carcinoma, using the developed method. The proteins from the microdissected cells were extracted, precipitated, digested with trypsin and then O-16/O-18 labeled. The normal and metastatic cell samples were analyzed using reversed phase LC-ESI-MS/MS on the ion trap mass spectrometer. A total of 76 proteins were identified. Some, such as mitochondrial isocitrate clehydrogenase, actin and 14-3-3 protein zeta/delta were found to be significantly up-regulated in the breast tumor cells.