Microtechnologies Enabling LCM-Based Clinical Proteomics

微技术支持基于 LCM 的临床蛋白质组学

• 批准号: 7125617
• 负责人: weijie wang
• 金额: $ 20万
• 依托单位: CALIBRANT BIOSYSTEMS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-25 至 2008-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7125617
• 关键词: Microtechnologies; Enabling; LCM; Based; Clinical

DESCRIPTION (provided by applicant): Proteomic analysis of laser capture microdissection (LCM) procured specimens is severely constrained by sample amounts ranging from 1,000-100,000 cells, corresponding to a total protein content of 0.1-10 microgram. Current proteome technologies, including two-dimensional (2D) polyacrylamide gel electrophoresis and shotgun-based multidimensional liquid chromatography separations, require large cellular samples that are orders of magnitude greater than those obtained during a clinical biopsy. Thus, this project aims to develop and demonstrate a capillary gel electrophoresis (CGE)-based multidimensional separation platform, capable of performing comprehensive and ultrasensitive studies of protein profiles within LCM procured specimens. A key feature of the proposed proteome technology is the simplification of many of the common sample handling steps, as the tissue sample acquired through LCM is directly processed and applied to the inlet end of the CGE capillary through electrokinetic injection and stacking of SDS-protein complexes. This feature allows the quantitative use of limited protein samples by combining analyte concentration, protein/peptide separations, and in situ proteolytic digestion in an integrated platform while eliminating analyte loss and dilution to achieve comprehensive and ultrasensitive proteomic studies. Several performance factors in the proposed multidimensional separation platform equipped with ESI-qTOF MS, including the protein concentration coefficient of at least 500-fold contributed by electrokinetic stacking, the dynamic range (the ratio of high abundance to low abundance proteins) of 100,000: 1 or higher, and the overall peak capacity of more than 70,000, will be evaluated using yeast cell lysates containing model proteins such as ribonuclease A, casein, and green fluorescence protein of known concentrations. The analytical capabilities of the proposed proteome technology will be demonstrated using LCM procured tissue specimens obtained from Professor Wittliff's laboratory at the University of Louisville. The respective scientific milestones include the use of 1 microgram or less total protein loading for enabling the identification of at least 1,000 proteins from tissue specimens with greater than 80% reproducibility in identified proteins among replicates.

描述（由申请人提供）：激光捕获显微解剖（LCM）获得的样品的蛋白质组学分析受到样品数量的严格限制，样品数量范围为1,000-100,000个细胞，对应于总蛋白质含量为0.1-10微克。目前的蛋白质组技术，包括二维（2D）聚丙烯酰胺凝胶电泳和基于散弹枪的多维液相色谱分离，需要比临床活检获得的大几个数量级的大细胞样本。因此，该项目旨在开发和演示基于毛细管凝胶电泳（CGE）的多维分离平台，能够对LCM获得的标本进行全面和超灵敏的蛋白质谱研究。所提出的蛋白质组技术的一个关键特征是简化了许多常见的样品处理步骤，因为通过LCM获得的组织样品直接处理，并通过电动注射和sds -蛋白质复合物的堆叠应用于CGE毛细管的入口端。该功能允许定量使用有限的蛋白质样品，通过将分析物浓度，蛋白质/肽分离和原位蛋白水解消化结合在一个集成平台上，同时消除分析物损失和稀释，实现全面和超灵敏的蛋白质组学研究。所提出的ESI-qTOF质谱多维分离平台的几个性能因素包括：蛋白质浓度系数至少为电动叠加贡献的500倍，动态范围（高丰度与低丰度蛋白质之比）为100,000；1或更高，并且总峰值容量超过70,000，将使用含有模型蛋白（如核糖核酸酶A，酪蛋白和已知浓度的绿色荧光蛋白）的酵母细胞裂解液进行评估。提出的蛋白质组技术的分析能力将使用LCM从路易斯维尔大学Wittliff教授实验室获得的组织标本来证明。相应的科学里程碑包括使用1微克或更少的总蛋白质负载，能够从组织标本中鉴定至少1000种蛋白质，在所鉴定的蛋白质中重复的可重复性大于80%。