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

开发用于定量分析的无损分子提取平台

• 批准号: 8754932
• 负责人: Brian M Balgley
• 金额: $ 15万
• 依托单位: BIO-QUICK CORPORATION
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8754932
• 关键词: Abnormal Cell; Affect; Amino Acids; Antigens; Archives; Biological Assay; Biopsy; Buffers; Businesses; Cell Culture Techniques; Cell Extracts; Cell Line; Cells; Complement; Core Biopsy; Data; Development; Devices; Digestion; Disease; Early Diagnosis; Formalin; Funding; Goals; Hematoxylin and Eosin Staining Method; Histopathology; Immunohistochemistry; In Situ Hybridization; Lasers; Lesion; Liquid Chromatography; Malignant Neoplasms; Mammary Neoplasms; Mass Spectrum Analysis; Measurement; Measures; Methods; Microscope; Mind; Molecular; Molecular Analysis; Morphology; National Institute of Environmental Health Sciences; Nucleic Acids; Nucleotides; Paraffin Embedding; Pathologist; Patients; Peptides; Phase; Phenotype; Population; Process; Proteins; Proteome; Proteomics; Protocols documentation; Quality Control; Reference Standards; Relative (related person); Request for Applications; Retrieval; Sampling; Shotguns; Slide; Solutions; Specimen; Stable Isotope Labeling; Staining method; Stains; Standardization; System; Techniques; Testing; Time; Tissue Sample; Tissues; Tumor Cell Line; Tumor Subtype; United States National Institutes of Health; Variant; Work; base; high standard; improved; instrumentation; interest; novel; protein profiling; public health relevance; response; sample fixation; tandem mass spectrometry; tissue fixing; tumor

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.

描述（由申请人提供）：在本研究中，我们提出了一个非破坏性分子提取 (NDME) 平台，用于使用 SILAC 细胞点对照对 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 后剩余的组织切片将用于组织病理学分析，例如 H&E 染色、多重免疫组织化学染色和原位杂交。 SILAC 点对照载玻片由使用稳定同位素标记氨基酸 (SILAC) 生长的细胞培养物制备而成，经过福尔马林固定和石蜡包埋，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均等量存在，因此组织样品和 SILAC 细胞点对照之间的肽倍数变化比可用于对大量样品进行定量分析。因此，它们可以提供蛋白质组学测量的标准化。此外，由于 NDME 在载玻片上进行，因此它提供了组织载玻片宏观解剖的可能性，即从周围正常/非肿瘤背景中分离肿瘤/异常细胞及其前体病变，提取来自异质组织的兴趣，以丰富与疾病相关的蛋白质并提高测定的灵敏度。在此一期申请中，我们寻求资金以：1) 优化 NDME 提取缓冲液和方案，以实现下游鸟枪法蛋白质组学分析的有效提取，以及 2) 开发和评估存档的定量鸟枪法蛋白质组学研究的一系列 SILAC 细胞点标准品乳腺肿瘤 FFPE 组织。