Ultrasensitive Approaches for Complex Animal Proteomics

描述（由申请方提供）：只有当研究的器官或组织样本由同质细胞群组成时，才能生成生物学和临床相关蛋白质组学数据，其中没有不同类型和/或发育阶段的多余细胞会掩盖结果。组织样品分析的主要问题之一是样品的异质性。许多不同的细胞类型通常存在于组织活检中，并且在患病组织的情况下，少量异常细胞可能位于未受影响的区域内或邻近未受影响的区域。因此，激光捕获/显微切割过程已被开发，以提供一种快速和直接的方法，用于获得均匀的细胞亚群或结构，用于生物化学和分子生物学分析。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chromatofocusing，固定化pH膜，Rotofor，自由流动电泳，离凝胶电泳，都是在电泳规模和不兼容的小细胞群体收集从显微解剖采购标本。因此，我们的研究目标是将Gemini蛋白质组学平台与组织显微切割/制备技术相结合，作为一种新的生物标志物发现范式，用于对从临床组织标本中分离的靶细胞群中的蛋白质表达谱进行全面和比较调查。所提出的生物标志物发现范例的开发、评估和验证的明显进展包括（i）使用多路复用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能力，以比较表征胶质瘤恶性肿瘤的显微切割肿瘤细胞内的蛋白质表达谱，和（iii）在大量其他组织样本上使用蛋白质印迹和免疫组织化学技术确认不同级别的胶质瘤恶性肿瘤之间的区别性生物标志物候选物。除了临床蛋白质组学技术的开发和验证，第二阶段的工作还将集中在疾病标志物的鉴定，不同疾病状态和表型之间的分子关系的探索，以及对推动疾病进展的分子机制的更深入的理解。 公共卫生相关性：我们的研究目标是将第一阶段项目中出现的Gemini蛋白质组学平台与组织显微切割/制备技术相结合，作为一种新的生物标志物发现范式，用于对从临床组织标本中分离的靶细胞群中的蛋白质表达谱进行全面和比较调查。使用来自高质量组织标本的富集恶性细胞的靶向蛋白质组学研究的最大期望在于在临床环境中鉴定诊断、预后和预测生物标志物，以及在生物制药行业中发现和验证新的蛋白质靶点。

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.