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DIA analysis of cellular reprogramming using SWATH-MS
[Thesis]. Manchester, UK: The University of Manchester; 2019.
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Abstract
Differentiated cells can be reprogrammed into a pluripotent state by forced expression of the Oct4, Sox2, c-Myc and Klf4 transcription factors (OKSM), thus generating induced pluripotent stem cell (iPSC) lines. IPSC lines can produce an impressive range of cell types in the organism and are therefore similar to embryonic stem cells (ESCs). However, iPSCs offer a unique opportunity for patient-specific cell replacement therapies, disease modelling and drug discovery. The generation of iPSCs however, is largely inefficient and slow, which complicates mechanistic studies. Here, ColSC/M2rtTA/Oct4-GFP mouse embryonic fibroblasts containing the OKSM factors in a doxycycline-inducible lentiviral vector were used to further understand reprogramming using proteomics. Fourteen different reprogramming populations between days 0 and 20 were collected by flow cytometric sorting. Following the down-regulation of Thy1 by day 3, up-regulation of SSEA1 by day 6 and activation of Oct4-GFP beyond day 9 was observed. Cell sorting based on the expression of these markers showed significant enrichment of cell populations and gradual downregulation of fibroblast markers expression with up-regulated stem cell markers expression noted by qPCR analysis. A novel, in-house mouse protein library was generated prior to the SWATH mass spectrometry (MS) analysis containing 7,740 proteins including ESC markers of relatively low abundance, Oct4, Nanog, Esrrb and Fbx15, showing its high quality and coverage. The spectral information from the protein library was matched to the complex information recorded in the permanent digital SWATH maps derived from reprogramming cell samples by targeted data extraction. In total, 5,154 unique proteins were confidently identified across all samples using the Stoller Centre bioinformatics pipeline and 4,525 proteins using Markerview software. Data analysis showed that multiple functionally related proteins changed in expression in a highly coordinated manner. These included increased cell proliferation and metabolism (e.g. glycolysis) proteins and down-regulation of cell adhesion, protein transport and lysosomal proteins as early as day 3. The analysis showed that the major reorganisation of the proteome occurred as early as day 3. It was between days 10 and 13 where the large majority of proteins were altered in expression. The Markerview software platform showed similar reorganisation of the proteome, however, Markerview analysis showed a larger ratio of down-regulated proteins to up-regulated proteins in comparison to the SBDC analysis. In addition, PDGFRA and YES1 cell surface proteins showed a gradual upregulation and may have value as markers in reprogramming development.