Ultrasensitive Approaches for Complex Animal Proteomics

描述（由申请人提供）：生物学和临床相关的蛋白质组学数据只能在被调查的器官或组织样本由均匀的细胞群组成，其中没有不同类型和/或发育阶段的不需要的细胞掩盖结果的情况下产生。组织样品分析的主要问题之一是样品的异质性。在组织活检中通常存在许多不同的细胞类型，在病变组织的情况下，少量异常细胞可能位于未受影响的区域内或附近。因此，激光捕获/微解剖过程已经发展为提供一种快速和直接的方法来获取均匀的细胞亚群或结构进行生化和分子生物学分析。然而，目前的蛋白质组学技术，包括二维聚丙烯酰胺凝胶电泳、多维液相色谱系统、凝胶和无凝胶等电聚焦方法，如色谱聚焦、固定化pH膜、Rotofor、自由流动电泳和非凝胶电泳，都是在制备规模上操作的，与从微解剖获得的标本中收集的小细胞群不相容。因此，我们的研究目标是将Gemini蛋白质组学平台与组织显微解剖/制备技术结合起来，作为一种新的生物标志物发现范例，用于从临床组织标本中分离的靶向细胞群中进行蛋白质表达谱的全面和比较调查。在开发、评估和验证所提出的生物标志物发现范式方面的明显进展包括：(i)使用多路纳米反相液相色谱系统实现高通量Gemini蛋白质组学平台；（ii）全面评估Calibrant独特的组织蛋白质组学能力，以比较表征恶性胶质瘤微解剖肿瘤细胞中的蛋白质表达谱；（iii）利用Western blot和免疫组织化学技术，在大量额外的组织标本上，确认不同级别的胶质瘤恶性肿瘤中区分候选生物标志物。除了临床蛋白质组学技术的开发和验证外，II期的工作还将侧重于疾病标志物的鉴定，探索不同疾病状态和表型之间的分子关系，以及更深入地了解驱动疾病进展的分子机制。

DESCRIPTION (provided by applicant): Biologically and clinically relevant proteomic data can only be generated if organ or tissue samples investigated consist of homogeneous cell populations, in which no unwanted cells of different types and/or development stages obscure the results. One of the main problems with the analysis of tissue samples is the heterogeneous nature of the sample. Many different cell types are typically present in tissue biopsies, and in the case of diseased tissue small numbers of abnormal cells may lie within or adjacent to unaffected areas. Thus, the laser capture/microdissection process has been developed to provide a rapid and straightforward method for procuring homogeneous subpopulations of cells or structures for biochemical and molecular biological analyses. However, current proteomic techniques, including two-dimensional polyacrylamide gel electrophoresis, multidimensional liquid chromatography systems, and gel and gel-free isoelectric focusing approaches such as chromatofocusing, immobilized pH membranes, Rotofor, free-flow electrophoresis, and off-gel electrophoresis, are all operated at the preparative-scale and are incompatible with small cell populations collected from microdissection-procured specimens. Our research goal is therefore to integrate the Gemini proteomic platform with tissue microdissection/preparation techniques as a novel biomarker discovery paradigm for enabling comprehensive and comparative survey of protein expression profiles in targeted cell populations isolated from clinical tissue specimens. The obvious progression in the development, evaluation, and validation of proposed biomarker discovery paradigm includes (i) the implementation of high throughput Gemini proteomic platform using a multiplexed nano-reversed-phase liquid chromatography system, (ii) full assessment of Calibrant's unique tissue proteome capabilities toward comparative characterization of protein expression profiles within microdissected tumor cells of glioma malignancies, and (iii) confirmation of discriminate biomarker candidates among different grades of glioma malignancies using Western blot and immunohistochemistry techniques on a large series of additional tissue specimens. In addition to the clinical proteomic technology development and validation, Phase II efforts will also focus on the identification of disease markers, exploration of molecular relationships among different disease states and phenotypes, and a deeper understanding of molecular mechanisms that drive disease progression. PUBLIC HEALTH RELEVANCE: Our research goal is to integrate the Gemini proteomic platform emerging from the Phase I project with tissue microdissection/preparation techniques as a novel biomarker discovery paradigm for enabling comprehensive and comparative survey of protein expression profiles in targeted cell populations isolated from clinical tissue specimens. The greatest expectations for targeted proteomic research using enriched malignant cells from high quality tissue specimens reside in the identification of diagnostic, prognostic, and predictive biological markers in the clinical setting, as well as the discovery and validation of new protein targets in the biopharmaceutical industry.