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Design, development and application of new technological approaches in subcellular proteomics

McGill University
Publication Date
  • Biology - Cell
  • Biology
  • Chemistry
  • Computer Science
  • Design
  • Medicine


The field of subcellular proteomics aims to describe and analyze all the proteins present in a precise subcellular compartment at a given time. In contrast to whole-cell or whole-organism proteomics, the analysis of individual organelles has provided simpler proteomes from which relevant biological information could be more easily derived. To date, the protein complement of several subcellular structures, including the mitochondria, lysosome, peroxysome, phagosome and nucleus has been described. The powerful and rapidly evolving instrumentation as well as the development of biochemical and bioinformatics tools now allow scientists to derive whole organelle models based on the proteomic data generated. This field however still faces numerous challenges. Among those is the analysis of membrane-associated proteins, whose large and hydrophobic character complicate their extraction, subsequent separation and analysis by mass spectrometry. Another emerging limitation in subcellular proteomic resides in the difficulty in collecting enough material of a very pure preparation of the organelle of interest, which still depend on lengthy and labor-intensive density-based centrifugation as the method of choice for subcellular fractionation. The work presented in this thesis describes the development of new methods for subcellular proteomics that address the above-mentioned limitations, and their application to relevant biological models. In chapter 2, we present the design of a non-discriminatory investigative approach to study membrane proteins. Relying on detergent-free and gel-free procedures, this strategy allowed identification of hundreds of cell surface-exposed proteins from freshly ejaculated bovine spermatozoa, as presented in chapter 3. Diverging from traditional cell fractionation protocols, we have refined in chapter 4 a fluorescence-assisted organelle sorting method and have employed it to acquire the proteome of corticotropes-derived secretory granules. Our proc

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