Development of a non-destructive molecular extraction platform for quantitative p

描述(由申请人提供)：在这项研究中，我们提出了一种非破坏性分子提取(NDME)平台，以SILAC细胞点为对照对FFPE样本进行定量蛋白质组学分析。NDME技术可以直接从单个固定的组织切片中提取蛋白质，而不破坏组织形态。NDME后残留的组织切片将用于组织病理学分析，如H&E染色、多重免疫组织化学染色和原位杂交。用稳定同位素标记氨基酸(SILAC)培养的细胞经福尔马林固定和石蜡包埋后制备SILAC斑点对照玻片，将与组织标本一起进行NDME。由于SILAC细胞点对照在所有情况下都是等量存在的，因此组织样本和SILAC细胞点对照之间的多肽折叠变化率可用于对大量样本进行定量分析。因此，它们可以提供蛋白质组学测量的标准化。此外，由于NDME是在载玻片上进行的，它提供了在载玻片上对组织进行宏观解剖的可能性，即从周围的正常/非肿瘤背景中分离出肿瘤/异常细胞及其前驱病变，从不同的组织中提取感兴趣的细胞群，以丰富与疾病相关的蛋白并提高分析的灵敏度。在这一第一阶段的应用中，我们寻求资金用于：1)优化NDME提取缓冲液和协议，以实现用于下游鸟枪式蛋白质组分析的有效提取，以及2)开发和评估一系列SILAC细胞点标准，用于对存档的乳腺肿瘤FFPE组织进行定量鸟枪式蛋白质组学研究。

DESCRIPTION (provided by applicant): In this study, we propose a Non-Destructive Molecular Extraction (NDME) platform to perform quantitative proteomics analysis of FFPE specimens with SILAC cell dot controls. The NDME technique can directly extract proteins from a single fixed tissue section without destroying tissue morphology. The tissue section remaining after NDME will be used for histopathological analyses, such as H&E staining, multiplex immunohistochemistry staining, and in situ hybridization. The SILAC dot control slides, which are prepared from cell cultures grown using stable isotope labeled amino acids (SILAC) which have undergone formalin fixation and paraffin embedding, will undergo NDME together with the tissue specimens. Since the SILAC cell dot control is present in equal quantity in all cases, the peptide fold change ratio between the tissue samples and the SILAC cell dot control can be used for quantitative analysis across a large number of samples. Thus, they can provide standardization in proteomics measurements. In addition, since NDME is performed on slide, it offers the possibility for on-slide macrodissection of the tissue, i.e., for isolation of tumor/abnormal cells and their precursor lesions from surrounding normal/non-tumor background, extracting the cell population of interest from a heterogeneous tissue, to enrich the disease-relevant proteins and increase the sensitivity of the assay. In this Phase I application, we seek funding to: 1) optimize the NDME extraction buffers and protocols to achieve effective extractions for downstream shotgun proteomics analysis, and 2) develop and evaluate an array of SILAC cell dot standards for quantitative shotgun proteomics studies in archived breast tumor FFPE tissues.