Title Information
Title
Quantitative Phosphosproteomics Analysis of Basal T Cell Signaling Pathways and Optimized Methodology to Improve Proteomics Sequencing Depth
Name: Personal
Name Part
Chen, Zhuo
Role
Role Term: Text
creator
Origin Information
Copyright Date
2016
Physical Description
Extent
16, 116 p.
digitalOrigin
born digital
Note
Thesis (Ph.D. -- Brown University (2016)
Name: Personal
Name Part
Salomon, Arthur
Role
Role Term: Text
Director
Name: Personal
Name Part
Cane, David
Role
Role Term: Text
Reader
Name: Personal
Name Part
Peti, Wolfgang
Role
Role Term: Text
Reader
Name: Corporate
Name Part
Brown University. Chemistry
Role
Role Term: Text
sponsor
Genre (aat)
theses
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%.
Subject
Topic
Basal T cell signaling
Subject
Topic
Csk
Subject
Topic
CD45
Subject
Topic
Fritless column
Subject
Topic
Single-run proteomics
Subject
Topic
DMSO
Record Information
Record Content Source (marcorg)
RPB
Record Creation Date (encoding="iso8601")
20160629
Language
Language Term: Code (ISO639-2B)
eng
Language Term: Text
English
Identifier: DOI
10.7301/Z04B2ZP2
Access Condition: rights statement (href="http://rightsstatements.org/vocab/InC/1.0/")
In Copyright
Access Condition: restriction on access
Collection is open for research.
Type of Resource (primo)
dissertations