UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

A quantitative proteomics analysis of human cells undergoing apoptosis Anthony, Joseph Stephan


Elucidating the events and mechanism of regulation of apoptosis is of wide interest to the scientific community, and to humanity, since apoptosis, so important for proper development and maintenance of an organism, is also responsible for disease when the process goes awry. In this thesis, a proteomics investigation into changes in protein concentrations and half-lives in early apoptosis is presented, enhancing our understanding of this process. Using Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC), cytokine withdrawal-induced apoptosis of a human hematopoietic cell line, TF-1, was studied. This is a useful model in which signaling pathways regulating apoptosis have been extensively studied previously. A study such as this can be considered “hypothesis-generating”, but at the outset the hypothesis is that proteins whose functions are closely tied to regulation of apoptosis will show detectable changes in quantity in cells undergoing apoptosis. Initially three biological replicates were performed, comprising 200 samples in all, analyzed using an FT-ICR mass spectrometer. Relative abundance of 1451 proteins identified in common between three biological replicates was determined, and 124 proteins showing the largest concentration changes in response to cytokine withdrawal are discussed in more detail. A subsequent effort investigated protein half-life changes in response to cytokine withdrawal and identified 255 proteins for which half-lives were calculated. The apparent changes in protein half-life in response to cytokine withdrawal are discussed. A high level of coverage of the proteome was achieved, giving a large number of protein identifications and relative quantitations. Further I have been able to identify several apparently synchronous changes in concentration between proteins with related functions, suggesting possible interactions not previously described, or identified as playing a role in cell survival, proliferation or death. Further, I observed cytokine withdrawal-induced alterations in concentration in some proteins for which little is known. The proteomic analysis of apoptosis using SILAC to determine protein half-life data is also a novel approach. Together, the work in this thesis suggests numerous avenues of investigation potentially leading to novel findings regarding cells undergoing apoptosis; and also suggests a potentially fruitful avenue of investigation for clinical management of patients undergoing chemotherapy.

Item Media

Item Citations and Data


Attribution-NonCommercial-NoDerivs 3.0 Unported