Laser Capture Microscopy and 2D-DIGE: Cancer Proteomics

描述(由申请人提供)： 用传统方法分析肿瘤标本中蛋白质的障碍 包括准确量化的相对数量的困难 多个样品中的单个蛋白质和大多数固体的不均一性 肿瘤标本。这项提议通过将几个 跨专业协作努力中的新技术。这一战略 借鉴了临床医生、病理学家、基础科学家、 光谱学家和生物信息学家。为了获得相对的 均一群的肿瘤，癌细胞将被激光分离 捕获显微镜。虽然这项技术允许分离特定的 肿瘤内的细胞亚群，细胞总数往往较低。 因此，该提案将利用一种名为2-D Dge的新方法 (2维差示凝胶内电泳法)。在这种方法中，蛋白质 提取物在体外用一次荧光的活性菁染料进行标记。 几个选定的波长。最多三种提取物，标有不同的 染料在同一个大尺寸的二维凝胶中混合和分析。这个 凝胶在多个波长下成像并进行分析以确定准确的 蛋白质在不同部位迁移的比率。它的独特之处在于 技术是它允许独立定量来自于 同一凝胶中的两个或三个生物样本，消除了 凝胶到凝胶的重复性，因此提供了非常准确的 蛋白质组图谱。新一代染料允许检测和定量 以皮克量存在的单个多肽。通过将这一点结合起来 激光捕获显微切割敏感的蛋白质组检测方法 技术，应该有可能得到一个有用的蛋白质组图，从 低至1000至10,000个的细胞学同质标本 细胞。在这一阶段，我们将重点关注与癌症抗争的三个目标 激光捕获显微切割(LCM)获得的标本：直接比较 新一代半胱氨酸型活性染料与赖氨酸型活性染料 LCM样本的标签，以确定允许识别的策略 通过质谱学分析感兴趣的蛋白质种类，并进行试点 利用优化后的工艺对LCM和2D-DICE进行了实验。在下一个 阶段，将使用更大的样本来展示更广泛的临床应用 准备好了。蛋白质表达模式的变化将被识别出来。这些 改变可以作为临床上有用的肿瘤进展的标志物。

DESCRIPTION (provided by applicant): Barriers to the analysis of proteins in tumor samples by traditional methods include the difficulty in accurate quantitation of the relative amounts of individual proteins in multiple samples and the inhomogeneity of most solid tumor specimens. This proposal overcomes these barriers by combining several new technologies in a cross-specialty collaborative effort. This strategy draws upon expertise from clinicians, pathologists, basic scientists, mass spectroscopists, and bioinformaticists. In order to obtain relatively homogeneous populations of tumors, cancer cells will be isolated by laser capture microscopy. While this technique permits isolation of specific subpopulations of cells within tumors, the total number of cells is often low. The proposal will therefore draw on a newly developed approach called 2-D DIGE (2-Dimensional Differential In-Gel Electrophoresis). In this method, protein extracts are labeled in vitro with reactive cyanine dyes that fluoresce at one of several chosen wavelengths. Up to three extracts labeled with different dyes are mixed and analyzed in the same large-format two-dimensional gel. The gel is imaged at multiple wavelengths and analyzed to determine the precise ratio of proteins migrating in various spots. The unique aspect of the technology is that it allows independent quantitation of proteins derived from two or three biological samples in the same gel, eliminating issues of gel-to-gel reproducibility and thus providing an exceptionally accurate proteome map. A new generation of dyes allows detection and quantitation of individual polypeptides present in picogram amounts. By combining this sensitive proteomic detection method with laser capture microdissection technology, it should be possible to derive a useful proteome map derived from cytologically homogeneous specimens containing as few as 1,000 to 10,000 cells. In this phase, we will focus on three goals for working with cancer samples obtained by laser capture microdissection (LCM): to directly compare the new generation of cysteine-reactive dyes to the lysine-reactive dyes for the labeling of LCM samples, to identify strategies that permit identification of protein species of interest by mass spectrometry, and to perform pilot experiments of LCM and 2D-DICE using the optimized techniques. In the next phase, broader clinical utility will be demonstrated using a larger sample set. Changes in the pattern of protein expression will be identified. These changes can serve as clinically useful markers of tumor progression.