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，我们提出了一种非破坏性分子提取（NDME）平台，用于对具有SILAC细胞点对照的FFPE标本进行定量蛋白质组学分析。NDME技术可以直接从单个固定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，如H&E染色、多重免疫组织化学染色、原位杂交。由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制备的SILAC点控制载玻片将与组织标本一起进行NDME。由于SILAC细胞点控制在所有情况下均以等量存在，因此组织样品与SILAC细胞点控制之间的肽折叠变化比可用于跨大量样品的定量分析。因此，它们可以为蛋白质组学测量提供标准化。此外，由于NDME是在载玻片上进行的，因此它提供了在载玻片上对组织进行宏观解剖的可能性，即从周围正常/非肿瘤背景中分离肿瘤/异常细胞及其前体病变，从异质组织中提取感兴趣的细胞群，以丰富疾病相关蛋白并提高检测的灵敏度。在这个I期申请中，我们寻求资金用于：1)优化NDME提取缓冲液和方案，以实现下游猎枪蛋白组学分析的有效提取；2)开发和评估一系列SILAC细胞点标准，用于在存档的乳腺肿瘤FFPE组织中进行定量猎枪蛋白组学研究。