Description
- Abstract:
- Reversible protein phosphorylation plays a pivotal role in the regulation of cellular signaling pathways. Current approaches in phosphoproteomics focus on analysis of the global phosphoproteome in a single cellular state or of receptor stimulation time course experiments, often with a restricted number of time points. Although these studies have provided some insights into newly discovered phosphorylation sites that may be involved in pathways, they alone do not provide enough information to make precise predictions of the placement of individual phosphorylation events within a signaling pathway. Protein disruption and site-directed mutagenesis are essential to clearly define the precise biological roles of the hundreds of newly discovered phosphorylation sites uncovered in modern proteomics experiments. We have combined genetic analysis with quantitative proteomic methods and recently developed visual analysis tools to dissect the tyrosine phosphoproteome of isogenic Zap-70 tyrosine kinase null and reconstituted Jurkat T-cells. In our approach, label-free quantitation using normalization to co-purified phosphopeptide standards is applied to assemble high density temporal data within a single cell type, either Zap-70 null or reconstituted cells, providing a list of candidate phosphorylation sites that change in abundance after T-cell stimulation. SILAC ratios are then used to compare Zap-70 null and reconstituted cells across a time course of receptor stimulation, providing direct information about the placement of newly observed phosphorylation sites relative to Zap-70. These methods are adaptable to any cell culture signaling system in which isogenic wild type and mutant cells have been or can be derived using any available phosphopeptide enrichment strategy. These studies here have revealed quantitative phosphoproteomic signatures, which may be indicative of the ERK positive feedback pathway and Zap-70-dependent FYN regulation. Follow-up studies were performed and discussed in order to further examine these results. Finally, our group has engaged in our own method development, with the aim of improving our sample preparation procedure for LC/MS/MS analysis. A battery of experiments was performed on pervanadate-stimulated Jurkat T-cells and modifications to our sample preparation procedure were discussed.
- Notes:
- Thesis (Ph.D. -- Brown University (2011)
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Citation
Nguyen, Vinh Hai,
"A Quantitative Phosphoproteomic Analysis of the T-Cell Receptor Signaling Pathway Using Novel High-Throughput Approaches"
(2011).
Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.7301/Z04Q7S7V
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Molecular Biology, Cell Biology, and Biochemistry Theses and Dissertations
Theses and Dissertations for the Molecular Biology, Cell Biology, and Biochemistry department....