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.
This application is submitted in response to the request for Applications for Novel Methods for Obtaining Molecular Information from Archived Tissue Samples from NIEHS/NIH for small business concerns. One of the objectives of the announcement is devising standards for quantitation and normalization of molecular measures in FFPE tissues, including assessing the quality of proteins or nucleic acids in archived tissues stored for longer time periods. With that goal in mind, we propose development of a non-destructive molecule extraction platform for quantitative shotgun proteomics study of FFPE tissue in conjunction with a stable isotope labeled cell line standard.
