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dc.contributor.authorAngel, Peggi M.
dc.description.abstractEmbryonic stem cells are derived from the inner cell mass of the preimplantation embryo and are defined by properties of pluripotency and the ability to remain undifferentiated while proliferating in cell culture. When these cells are provoked towards spontaneous or directed differentiation, they provide investigative tools for studying the dynamic cellular mechanisms that occur during early development. While there are many reports of the transcriptional profiling of differentiating embryonic stem cells, there is little information on the translational events that occur during differentiation. In this work, we explore and develop methodologies for producing a quantitative description of translational events. We conclude by providing a comparative proteomic description of differentiating embryonic stem cells, applying the observed protein expression changes as an in vitro model of early embryogenesis. First, we investigate quantitation of a proteomic population using stable isotopic labeling (SIL) methods. Trypsin commonly is used to facilitate incorporation of two O atoms into the C-termini of the peptide population. We examine methods for preventing loss of the stable isotopes, a phenomenon that will hinder quantitative efforts. Removal of trypsin from the solution was found to be the best way to prevent loss of the label. In a related observation, we found that trypsin facilitated O incorporation has the potential to greatly increase the false discovery rate of sites of N-linked glycosylation. We demonstrate that this process occurs on an N-glycosylated standard protein and explore ways to prevent the problem. This problem was best solved by removal of trypsin from solution prior to removal of the N-linked glycosylate from the protein. We develop an alternative method for SIL using urea, a chaotrope used to denature and dissolve protein pellets. This method was comparable to other stable isotopic labeling strategies; however, the addition of the tag to C-terminal lysine residues decreased the intensity of the precursor ion during MALDI-TOF analysis. This method may prove useful for samples that require a strong chaotrope prior to proteolysis. Last, we use spectral counts, a simple label-free method, to comparatively describe the protein population of differentiating embryonic stem cells. A total of 1498 proteins were identified, 22% of which were predicted and hypothetical proteins. Regulated protein comprised 15% of the population. We discuss the identified protein expression comparative to known embryogenetic events.
dc.subject16O/18O labeling
dc.subjectN-linked glycosylation site mapping
dc.subjectstable isotopic labeling
dc.subjectembryonic stem cell
dc.titleQuantitative proteomics
dc.title.alternativemethodologies and comparative analysis of differentiating murine embryonic stem cells
dc.description.advisorRon Orlando
dc.description.committeeRon Orlando
dc.description.committeeLance Wells
dc.description.committeeJonathan Amster

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