With the ability to rapidly screen and manipulate genomes, the in depth study of the functional actors of biology—metabolites and proteins—is necessary to understand complex biochemistry in developmental and disease states. The analytical processes by which biological information is gained from metabolomics and proteomics experiments must also evolve with our understanding of biological complexity. Proteomic technologies have advanced in recent years to support more routine analyses of intact antibodies, enzymes and protein complexes. Additionally, breakthroughs in instrumentation and preanalytical techniques have allowed for increased speed and multiplexing to quantify peptides and more robustly characterize dynamic proteomic states and settings. Here, top-down proteomics, an approach that studies intact proteins, is used in conjunction with metabolomics to characterize the inhibition mechanism of covalent inhibitors of an aminotransferases with roles in cancer. Separately, moving past the routine study of intracellular proteins, quantitative top-down proteomics is also used to describe the complexity of proteins secreted from senescent cells. Quantitative tandem-mass tag based proteomics identified dynamic changes in the whole cell extract of cells undergoing cell cycle arrest due to senescence while top-down proteomics highlighted additional complexity in factors that reinforce cell cycle exit in senescent cells. Furthermore, quantitative metabolomics and carbon-13 flux analyses are used to demonstrate that senescent cells alter their metabolism to drive pyruvate into the tricarboxylate acid (TCA) cycle and do not metabolize glutamine to the same extent as growing cells, even though they express mutant, oncogenic HRAS. Beyond the study of senescence, top-down and bottom-up proteomics are used to quantitatively study primary colorectal adenocarcinomas and colorectal cancer cells. A comparative analysis of nine primary tumors highlights the heterogeneity of colorectal cancer at the proteomic level. An investigation of colorectal cancer cells isogenic for mutant KRAS identified upregulation of several urea cycle and aspartate metabolizing proteins. Several of the upregulated proteins, including asparagine synthetase and phosphoenolpyruvate carboxykinase have been previously identified to support colorectal cancer cell growth. Argininosuccinate synthase is examined in more detail across clinical datasets and through the establishment of doxycycline-inducible knock-out cell lines. These studies highlight the role of aspartate metabolism in colorectal cancer cell programs. Additionally, given the unique biochemistry of compartmentalized organelles within the cell, here a new transgene-based and inducible mitochondrial purification technique is established. Taking advantage of poly-histidine affinity reagents, the mitochondrial proteome is examined by top-down proteomics in melanoma cells. Together, these studies highlight top-down proteomics and multi-omics investigations for their ability to provide functional insight. Moreover, these advances demonstrate how more targeted studies can be used to assign functional significance provide mechanistic information.

Creator

• DOUBLEDAY, PETER

DOI

• https://doi.org/10.21985/n2-8xdm-at25

Subject

• Biology

Language

• en

Alternate Identifier

• http://dissertations.umi.com/northwestern:15013
• etdadmin_upload_719623

Date created

• 2020-01-01

Resource type

• Dissertation

Rights statement

• In Copyright