Laser Capture Microscopy and 2D-DIGE: Cancer Proteomics

描述（由申请人提供）： 传统方法分析肿瘤标本中蛋白质的障碍 包括难以准确定量 多个样品中的单个蛋白质和大多数固体样品的不均匀性 肿瘤标本该提案通过结合几个 跨专业协作的新技术。这一战略 从临床医生，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 肿瘤的同质群体，癌细胞将通过激光分离 捕获显微镜。虽然这种技术允许分离特定的 在肿瘤内的细胞亚群中，细胞总数通常很低。 因此，该提案将借鉴一种新开发的称为2-D DIGE的方法 （2-维差示凝胶内电泳）。在该方法中，蛋白质 提取物在体外用活性花青染料标记， 几个选定的波长。最多三种浸提液标记有不同的 染料在相同的大尺寸二维凝胶中混合和分析。的 凝胶在多个波长下成像并进行分析以确定 蛋白质在不同点迁移的比率。的独特之处 这项技术允许对来自于 两个或三个生物样品在同一凝胶中， 凝胶-凝胶重现性，从而提供了一个非常准确的 蛋白质组图谱新一代的染料可以检测和定量 以皮克量存在的单个多肽。通过组合该 激光捕获显微切割的蛋白质组学检测方法 技术，它应该是可能的，以获得一个有用的蛋白质组图来自 在细胞学上均质的标本中含有1,000到10000个 细胞在这个阶段，我们将专注于与癌症合作的三个目标 通过激光捕获显微切割（LCM）获得的样品：直接比较 新一代半胱氨酸活性染料以赖氨酸活性染料为 LCM样品的标记，以确定允许识别的策略 通过质谱法分析感兴趣的蛋白质种类，并进行试点 LCM和2D-DICE的实验使用优化的技术。未来 阶段，将使用更大的样本证明更广泛的临床实用性 集将鉴定蛋白质表达模式的变化。这些 这些变化可以作为肿瘤进展的临床有用标记。

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.