Embryonic stem cells (ESC) hold the promise to revolutionize reparative medicine through the development of stem cell-based therapies. This potential centers on the intrinsic abilities of proliferating ES cells to self-renew and to mature into essentially any cell type (pluripotency). Transplanting stem cells into damaged myocardium is emerging as a novel means for both acute repair and treatment of end-stage heart failure. The crux for therapeutic success will lie in being able to identify and manipulate proliferating ESCs to differentiate specifically into cardiac muscle upon demand. This proposal uses a series of synergistic and sophisticated proteomic technologies to address discrete biological and clinical questions with respect to ESC. In this discovery based grant, we focus on 4 distinctive sub-proteomes and include both downstream validation and functional analysis in order to develop new tools and provide insight into the regulation of ESC.
Specific aim 1 focuses on the identification of cell surface proteins, in particular the N-linked glycoproteome to create specific biomarker panel for the identification and monitoring of various stages of differentiation. Furthermore, we link the cell surface receptor identification with the secreted regulatory factors (e.g. paracrine factors) found during the same early stages of differentiation.
Specific aim 2 focuses on differential phospho-proteome analysis of ES cells over the initial 24 hours following differentiation initiated by serum starvation. This is the same time period in which the transcription factor, B-myb, is phosphorylated. B-myb is potentially one of the earliest triggers of differentiation.
Specific aim 3, investigates B-myb with respect to its regulation by phosphorylation, alterations in this protein complex and the functional consequences on the early stemness proteins (eg. Oct 3 and Nanog). Understanding the complex and dynamic inter-relationships within the ESC proteome is of basic biological interest, and has major clinical implications.
|Gundry, Rebekah L; Riordon, Daniel R; Tarasova, Yelena et al. (2012) A cell surfaceome map for immunophenotyping and sorting pluripotent stem cells. Mol Cell Proteomics 11:303-16|
|Gundry, Rebekah L; Tchernyshyov, Irina; Sheng, Shijun et al. (2010) Expanding the mouse embryonic stem cell proteome: combining three proteomic approaches. Proteomics 10:2728-32|
|Gao, Ping; Tchernyshyov, Irina; Chang, Tsung-Cheng et al. (2009) c-Myc suppression of miR-23a/b enhances mitochondrial glutaminase expression and glutamine metabolism. Nature 458:762-5|
|Gundry, Rebekah L; White, Melanie Y; Nogee, Julie et al. (2009) Assessment of albumin removal from an immunoaffinity spin column: critical implications for proteomic examination of the albuminome and albumin-depleted samples. Proteomics 9:2021-8|
|Stastna, Miroslava; Abraham, M Roselle; Van Eyk, Jennifer E (2009) Cardiac stem/progenitor cells, secreted proteins, and proteomics. FEBS Lett 583:1800-7|
|Gundry, Rebekah L; Raginski, Kimberly; Tarasova, Yelena et al. (2009) The mouse C2C12 myoblast cell surface N-linked glycoproteome: identification, glycosite occupancy, and membrane orientation. Mol Cell Proteomics 8:2555-69|
|Gundry, Rebekah L; White, Melanie Y; Murray, Christopher I et al. (2009) Preparation of proteins and peptides for mass spectrometry analysis in a bottom-up proteomics workflow. Curr Protoc Mol Biol Chapter 10:Unit10.25|
|Gundry, Rebekah L; Boheler, Kenneth R; Van Eyk, Jennifer E et al. (2008) A novel role for proteomics in the discovery of cell-surface markers on stem cells: Scratching the surface. Proteomics Clin Appl 2:892-903|
|Graham, David R M; Mitsak, Megan J; Elliott, Steven T et al. (2008) Two-dimensional gel-based approaches for the assessment of N-Linked and O-GlcNAc glycosylation in human and simian immunodeficiency viruses. Proteomics 8:4919-30|
|Yamanaka, Satoshi; Li, Jinliang; Kania, Gabriela et al. (2008) Pluripotency of embryonic stem cells. Cell Tissue Res 331:5-22|
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