Microtechnologies Enabling LCM-Based Clinical Proteomics

描述(由申请人提供)：激光捕获显微解剖(LCM)获得的样本的蛋白质组学分析受到从1,000-100,000个细胞的样本量的严格限制，对应于0.1-10微克的总蛋白质含量。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鸟枪的多维液相色谱分离，需要大量的细胞样本，这些样本比临床活检获得的样本大几个数量级。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获取的样品中的蛋白质图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主要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，并通过电动注入和堆积十二烷基硫酸钠-蛋白质复合体而被施加到毛细管电泳管的入口端。这一功能允许在一个集成平台中结合分析物浓缩、蛋白质/肽分离和原位蛋白质水解酶，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。配备ESI-qTOF MS的拟议多维分离平台中的几个性能因素，包括电动堆积贡献的至少500倍的蛋白质浓缩系数，100,000：1或更高的动态范围(高丰度与低丰度蛋白质的比率)，以及超过70,000的总峰容量，将使用包含已知浓度的核糖核酸酶A、酪蛋白和绿色荧光蛋白等模型蛋白质的酵母细胞裂解物进行评估。建议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教授的实验室获得的组织标本进行演示。各自的科学里程碑包括使用1微克或更少的总蛋白质负载量，以便能够从组织样本中鉴定至少1,000种蛋白质，在复制的蛋白质中识别的蛋白质的重复性大于80%。

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.