Identification of Differentially Expressed Proteomes in Human Astrocytomas

描述（由申请人提供）：生物学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，其中没有不同类型和/或发育阶段的不需要的细胞掩盖结果。对于组织活检的分析来说，这个问题更加复杂，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附近。虽然激光捕获显微解剖（LCM）等方法能够分离均匀的细胞亚群，但LCM获得的样品的蛋白质组学分析受到产生的样品数量非常少的严重限制。此外，已知LCM由于固定和染色过程而导致显著的蛋白质样品改变，进一步限制了从人类星形细胞瘤活检中进行有效蛋白质组学研究的能力。为了避免现有的蛋白质组技术在分析从显微解剖获得的组织标本中获得的蛋白质提取物时的局限性，最近在Calibrant开发了一个有效的基于发现的蛋白质组平台。这种名为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相结合，能够对通过组织显微解剖捕获的细胞中提取的微量蛋白质进行超灵敏分析。该项目进一步旨在采用由我们的合作者，国家神经疾病和中风研究所庄正平博士首创的一种新颖的无激光显微解剖技术，能够提供丰富，高质量和可重复的组织样本。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，将使用一组新鲜冷冻脑活检（每个肿瘤级别n = 4）进行，每个组织标本一式三份。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.