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: 6934914
• 负责人: DIYA REN
• 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 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6934914
• 关键词: bioengineering /biomedical engineering; biomedical equipment development; capillary electrophoresis; clinical research; gel electrophoresis; human tissue; laser capture microdissection; proteomics; technology /technique development

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）：激光捕获显微切割（LCM）获得的标本的蛋白质组学分析受到样本量范围为1，000 - 100，000个细胞（对应于0.1-10微克的总蛋白质含量）的严重限制。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霰弹枪的多维液相色谱分离，需要大的细胞样本，其数量级大于临床活检期间获得的。因此，本项目旨在开发和展示一种基于毛细管凝胶电泳（CGE）的多维分离平台，能够对LCM采购标本中的蛋白质谱进行全面和超灵敏的研究。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获得的组织样品被直接处理并通过SDS-蛋白质复合物的电动注射和堆叠应用于CGE毛细管的入口端。该功能允许通过在集成平台中结合分析物浓缩、蛋白质/肽分离和原位蛋白水解消化来定量使用有限的蛋白质样品，同时消除分析物损失和稀释，以实现全面和超灵敏的蛋白质组学研究。在所提出的配备ESI-qTOF MS的多维分离平台中的几个性能因素，包括由电动堆积贡献的至少500倍的蛋白质浓度系数，（高丰度与低丰度蛋白质的比率）为100，000：1或更高，以及大于70，000的总峰容量，将使用含有已知浓度的模型蛋白质如核糖核酸酶A、酪蛋白和绿色荧光蛋白的酵母细胞裂解物进行评价。拟议的蛋白质组技术的分析能力将证明使用LCM采购组织标本从教授Wittliff的实验室在路易斯维尔大学。各自的科学里程碑包括使用1微克或更少的总蛋白质加载量，以便能够从组织标本中鉴定至少1，000种蛋白质，并且重复鉴定的蛋白质的重现性大于80%。