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    Analysis of selected cell populations in tissues by MALDI MS

    Aerni, Hans Rudolf
    : https://etd.library.vanderbilt.edu/etd-07102011-213331
    http://hdl.handle.net/1803/12891
    : 2011-07-11

    Abstract

    New highly sensitive strategies for the analysis of proteins from selected cell populations in morphologically complex tissues have been developed. These workflows combine the single cell specificity of laser capture microdissection (LCM) for the enrichment of cells with newly developed on-chip processing protocols and detection of the resulting peptides by MALDI FT-ICR MS. Systematic optimization of all steps of the workflows was carried out. On-chip chemistry including antigen retrieval, protein extraction and digestion with trypsin was optimized enabling the detection of peptides from fewer than 20 and 100 cells from fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue respectively. Taking advantage of the high peak capacity and sensitivity of MALDI FT-ICR MS, reproducible detection of over 3000 peaks from as few as 100 cells could be demonstrated. Peptide profiles obtained from fresh frozen and FFPE tissue from the same animal showed similar peak patterns indicating that this method is useful for clinical marker discovery from FFPE tissue. The optimized methods were applied for the analysis of human FFPE prostate cancer tissue and profiling of medium spiny neurons (MSNs) in rat brain. For the prostate tissue, distinct peptide profiles that could distinguish between tumor and normal epithelial cells could be obtained. Profiling of the MSNs in rat striatum revealed significantly different peptide signatures between the neurons and nearby myelinated axons confirming that the method can distinguish between different cell populations. Indeed, the developed protocols provide exciting new opportunities for proteomic analysis in morphologically complex tissues with single cell type specificity and high sensitivity.
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