Laser Capture Microscopy and 2D-DIGE: Cancer Proteomics

激光捕获显微镜和 2D-​​DIGE：癌症蛋白质组学

• 批准号: 7124372
• 负责人: William S. Dynan
• 金额: $ 33.35万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-05-01 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7124372
• 关键词: bioengineering /biomedical engineering; biomarker; cell sorting; cervix neoplasms; colon neoplasms; computer data analysis; cysteine; fluorescent dye /probe; gel electrophoresis; gene expression; high throughput technology; human subject; human tissue; laser capture microdissection; lysine; mass spectrometry; neoplasm /cancer; neoplasm /cancer genetics; neoplastic cell; protein purification; proteomics; robotics; technology /technique development; tissue /cell culture

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.

描述（由申请人提供）： 传统方法分析肿瘤样品中蛋白质的障碍 包括准确定量相对量的困难 多个样品中的单个蛋白质以及大多数固体的不均匀性 肿瘤标本。该提案通过结合多种方式克服了这些障碍 跨专业协作的新技术。这个策略 借鉴了临床医生、病理学家、基础科学家、大众的专业知识 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。虽然该技术允许隔离特定的 肿瘤内的细胞亚群，细胞总数通常较低。 因此，该提案将利用一种新开发的方法，称为 2-D DIGE （二维微分凝胶内电泳）。在该方法中，蛋白质 提取物在体外用活性花青染料标记，该染料在 1 时发出荧光 几个选定的波长。最多三种带有不同标记的提取物 染料在相同的大幅面二维凝胶中混合和分析。这 凝胶在多个波长下成像并进行分析以确定精确的 蛋白质在不同点迁移的比例。其独特之处在于 技术的特点是它允许对源自的蛋白质进行独立定量 同一凝胶中的两个或三个生物样品，消除了问题 凝胶之间的重现性，从而提供异常准确的 蛋白质组图谱。新一代染料可以检测和定量 单个多肽以皮克量存在。通过结合这个 激光捕获显微切割的灵敏蛋白质组检测方法 技术，应该有可能获得有用的蛋白质组图谱 细胞学同质样本含有少至 1,000 至 10,000 个细胞 细胞。在此阶段，我们将重点关注癌症治疗的三个目标 通过激光捕获显微切割 (LCM) 获得的样品：直接比较 新一代半胱氨酸反应性染料到赖氨酸反应性染料 LCM 样品的标签，以确定允许识别的策略 通过质谱分析感兴趣的蛋白质种类，并进行试点 使用优化技术进行 LCM 和 2D-​​DICE 实验。在接下来的 阶段，将使用更大的样本来证明更广泛的临床实用性 放。将鉴定蛋白质表达模式的变化。这些 变化可以作为肿瘤进展的临床有用标志。