Breast Cancer Proteomic via Laser-Free Microdissection and Gemini Technologies

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使结果模糊。由于通常存在许多不同的细胞类型，并且少量的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，因此组织活检的分析问题复杂化。虽然诸如激光捕获显微切割（LCM）的方法能够分离同质的细胞亚群，但是LCM获得的标本的蛋白质组学分析受到所产生的非常少量的样品的严重限制。为了避免已建立的蛋白质组技术的局限性，用于分析从显微切割获得的组织标本中获得的蛋白质提取物，最近在Calibrant开发了一种有效的基于发现的蛋白质组平台。这个蛋白质组平台被称为Gemini，它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，并允许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。该项目还旨在采用由我们的合作者，国家神经疾病和中风研究所（NINDS）的Zhengping Zhuang博士开创的新型无激光显微切割技术，能够提供丰富，高质量和可重复的组织样本。通过将Calibrant从微小样本中进行蛋白质组分析的能力与Zhuang博士（NINDS）在组织显微切割和肿瘤病理学方面提供的技术和专业知识相结合，拟议的研究代表了一种协同努力，旨在评估和验证一种新的生物标志物发现范式，以支持癌症研究，诊断和治疗的癌细胞及其微环境的蛋白质组分析。将通过与Fattaneh A教授的合作，实现在全球水平上研究与乳腺癌相关的分子机制的生物标志物发现平台的应用。Tavassoli（耶鲁大学医学院），他将应用30多年的乳腺癌病理学和生物学研究经验，并提供一系列新鲜冷冻的人类乳腺癌活检标本，用于生物标志物的发现。

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. 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 allows 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 (NINDS), capable of providing enriched, high quality, and reproducible tissue samples. By combining Calibrant's ability to perform proteomic profiling from minute samples with the technology and expertise offered by Dr. Zhuang (NINDS) in tissue microdissection and tumor pathology, the proposed research represents a synergistic effort toward the evaluation and validation of a novel biomarker discovery paradigm for enabling the proteome analysis of cancer cells and their micro-environment in support of cancer research, diagnosis, and treatment. Application of the resulting biomarker discovery platform for studying the molecular mechanisms associated with breast carcinoma at the global level will be realized through a collaboration with Professor Fattaneh A. Tavassoli (Yale University School of Medicine), who will apply more than 30 years of research experience in breast cancer pathology and biology and provide access to a collection of fresh frozen human breast cancer biopsies for biomarker discovery.