Identification of Differentially Expressed Proteomes in Human Astrocytomas

描述(由申请人提供)：生物相关蛋白质组学数据的生成需要由同质细胞群组成的样本，其中不同类型和/或发育阶段的不需要的细胞不会掩盖结果。对于组织活检的分析，问题更加复杂，因为通常存在许多不同的细胞类型，并且少量异常细胞可能位于未受影响的区域内或邻近区域。虽然激光捕获显微切割(LCM)等方法能够分离出同质的细胞亚群，但LCM获取的样本的蛋白质组学分析受到产生的样本量非常少的严重限制。此外，众所周知，由于固定和染色程序，LCM会导致蛋白质样本发生显著变化，进一步限制了从人类星形细胞瘤活检组织中进行有效蛋白质组研究的能力。为了避免已建立的蛋白质组技术在分析从显微解剖获取的组织标本中获得的蛋白质提取物方面的局限性，最近在CALIBRANT开发了一个有效的基于发现的蛋白质组平台。这个名为Gemini的蛋白质组平台将独特的多维分离系统与定制的后端生物信息学工具结合在一起，能够对从组织显微切割捕获的细胞中提取的微量蛋白质进行超灵敏分析。该项目还旨在采用我们的合作者、国家神经疾病和中风研究所的庄正平博士开创的一种新颖的、无激光的显微解剖技术，能够提供丰富、高质量和可重复的组织样本。对恶性胶质瘤的蛋白质组分析，包括II级星形细胞瘤、III级间变性星形细胞瘤和IV级多形性胶质母细胞瘤，将使用一套新鲜的冰冻脑活检组织(每个肿瘤级别n=4)，每个组织样本三份。比较蛋白质组学研究的结果有望揭示(I)相关的肿瘤相关生物标志物和网络，(Ii)不同星形细胞瘤之间的分子关系，以及(Iii)驱动星形细胞瘤进展的分子机制，例如II级到III级。

DESCRIPTION (provided by applicant): The generation of biologically relevant proteomics data requires samples consisting of homogenous cell populations, in which no unwanted cells of different types and/or development stages obscure the results. The problem is compounded for the analysis of tissue biopsies, since many different cell types are typically present, and small numbers of abnormal cells may lie within or adjacent to unaffected areas. While methods such as laser capture microdissection (LCM) enable the isolation of homogeneous subpopulations of cells, proteomic analysis of LCM-procured specimens is severely constrained by the very low amounts of sample generated. Furthermore, LCM is known to result in significant protein sample alterations due to fixation and staining procedures, further limiting the ability to perform effective proteome studies from human astrocytoma biopsies. To avoid the limitations of established proteome techniques for analyzing protein extracts obtained from microdissection-procured tissue specimens, an effective discovery-based proteome platform has recently been developed at Calibrant. This proteome platform, called Gemini, combines a unique multidimensional separation system with customized back-end bioinformatics tools, and enables ultrasensitive analysis of minute protein amounts extracted from cells captured by tissue microdissection. This project further aims to employ a novel, laser-free microdissection technique pioneered by our collaborator, Dr. Zhengping Zhuang at the National Institute of Neurological Disorders and Stroke, capable of providing enriched, high quality, and reproducible tissue samples. Proteome analysis of glioma malignancies, including grade II astrocytoma, grade III anaplastic astrocytoma, and grade IV glioblastoma multiforme, will be performed using a set of fresh frozen brain biopsies (n = 4 for each tumor grade) and in triplicates for each tissue specimen. The results of comparative proteomics studies are expected to reveal (i) relevant tumor-associated biological markers and networks, (ii) molecular relationships between different astrocytomas, and (iii) the molecular mechanisms that drive the progression of astrocytomas, e.g. grade II to grade III.