PROTEOME ANALYSIS OF FORMALIN-FIXED AND PARAFFIN-EMBEDDED TISSUE
Lee, Cheng S
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Because of the long history of the use of formalin as the standard fixative for tissue processing in histopathology, these archival formalin-fixed and paraffin-embedded (FFPE) tissues present invaluable resources for conducting retrospective disease investigations. However, the high degree of covalently cross-linked proteins in FFPE tissues hinders efficient extraction of proteins from tissue sections and prevents subsequent proteomics efforts from opening the door to a veritable treasure trove of information sequestered in archival tissues. To this end, a protein extraction methodology has been optimized and demonstrated to achieve effective protein extraction together with combined technological development for enabling comprehensive and comparative proteome studies across archival FFPE tissue collections. An effective discovery-based proteome platform combining capillary isoelectric focusing (CIEF)-based multidimensional separation system with electrospray ionization-mass spectrometry (ESI-MS) has been developed to enable ultrasensitive analysis of minute protein amounts extracted from targeted cells in tissue specimens in this thesis. Based on our initial success in analyzing protein profiles within microdissected FFPE tissues, this project further demonstrates the ability to achieve high confidence and comparative proteomic analysis using tissue blocks stored for as many as 28 years. Vacuolar proton translocating ATPase 116 kDa subunit isoform a3, one of the unique proteins expressed in the ASPS, is further validated by immunohistochemistry (IHC). Although IHC is highly sensitive and provides the subcellular resolution, MS-based proteome profiling enables global identification and quantification of thousands of proteins without a prior knowledge of individual proteins being analyzed or the need of validated antibodies.