Proteome turnover, the process by which proteins are continuously synthesized and degraded, is a crucial biological process for gene expression regulation, cell state maintenance, cellular homeostasis, and response to stimuli. This dissertation outlines novel methods to quantify proteome turnover in both mouse-derived organoid disease models and in vivo using stable isotope labeling combined with mass spectrometry-based methods.
In Chapter 1, we review recent LC-MS/MS techniques for measuring proteome turnover, highlighting their applications and limitations.
Chapter 2 focuses on a systematic analysis of proteome turnover in an organoid model of pancreatic ductal adenocarcinoma (PDA) using dynamic Stable Isotope Labeling of Organoids (dSILO). This study reveals faster proteome turnover in metastatic organoids compared to primary tumors and identifies several differentially regulated protein complexes in metastatic tumors, particularly from the mitochondrial respiratory chain.
In Chapter 3, we present the exploration of various methods for quantifying in vivo proteome turnover in mice. We employed an isotopic pulse-labeling strategy, dynamic Stable Isotopic Labeling of Mammals (dSILAM), then compared four combinations of mass spectrometry-based data acquisition and half-life modeling methods. We uncovered moderate differences in coverage, reproducibility, and half-life estimations between datasets, although further optimization is required for robust conclusions.
Chapter 4 discusses potential limitations and future directions for all of the work described herein.
Overall, our findings contribute to the optimization of proteomic workflows for studying protein turnover, which can be applied to enhance our understanding of cellular physiology and the molecular mechanisms underlying disease.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/xntg-xt15 |
| Date | January 2024 |
| Creators | Ross, Alison B. |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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