Quantitative Phosphosproteomics Analysis of Basal T Cell Signaling Pathways and Optimized Methodology to Improve Proteomics Sequencing Depth

Chen, Zhuo

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Year:: 2016
Contributor:: Chen, Zhuo (creator); Salomon, Arthur (Director); Cane, David (Reader); Peti, Wolfgang (Reader); Brown University. Chemistry (sponsor)
Genre:: theses
Subject:: Basal T cell signaling; Csk; CD45; Fritless column; Single-run proteomics; DMSO
Extent:: 16, 116 p.
DOI: https://doi.org/10.7301/Z04B2ZP2

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Abstract:: The basal T cell signaling is important in the T cells maturation and differentiation. The kinase-phosphatase pair Csk and CD45 regulate the basal signaling by modulating the activity of Src family kinases Lck and Fyn. In this study, we deployed a mass spectrometry-based quantitative phosphoproteomic approach to identify the signaling through Csk and CD45 during T cell basal signaling by comparing the wide-scale phosphorylation patterns in Csk and CD45 singly or doubly deficient cell lines with wild type Jurkat T cells. The results reveal that signaling cascades and cytoskeletal dynamics in the basal state of T cells share components with conventional TCR signaling. We also propose a new model of negative regulation of Fyn SH2 domain phosphorylation (on Y185, Y213, Y214) by CD45. Furthermore, based on the many hyperphosphorylated tyrosine sites detected in Csk and CD45 doubly deficient cells, we propose a synergistic regulation model of Lck kinase loop and Fyn SH2 domain, which regulate integrin-mediated signaling and cytoskeletal dynamics in T cells. The wide-scale adoption of sub-2 µm particles in HPLC columns has been hampered by the necessity for ultra-high pressure liquid chromatography or a column heating apparatus. We introduce a new strategy to fabricate a 50 cm-long, 1.9 µm particle C18 column, which was packed under 100 Bar and routinely operated below 300 Bar. Compared with 3 µm particles, the column with the 1.9 µm particles could detect 330% more peptides with statistically significant changes from differentially stimulated T cells. We thus provide an inexpensive improvement for single-run LC-MS/MS analysis to optimize sequencing depth, dynamic range, sensitivity, and reproducibility. This study also highlights the importance of the statistical analysis of quantitative proteomic data instead of a sole focus on peptides identification yields. Another effort is to optimize the protocol for the use of DMSO cosolvent with Q Exactive mass spectrometer. DMSO cosolvent was reported to improve the number of peptide identification with Orbitrap, but was found to contaminate Q Exactive. Our proposed protocol minimized the contamination while still increasing peptide identification by at least 10%.
Notes:: Thesis (Ph.D. -- Brown University (2016)

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Chen, Zhuo, "Quantitative Phosphosproteomics Analysis of Basal T Cell Signaling Pathways and Optimized Methodology to Improve Proteomics Sequencing Depth" (2016). Chemistry Theses and Dissertations. Brown Digital Repository. Brown University Library. https://doi.org/10.7301/Z04B2ZP2

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Chemistry Theses and Dissertations

Theses and Dissertations for the Chemistry department.
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