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Please use this identifier to cite or link to this item: http://arks.princeton.edu/ark:/88435/dsp01qf85nf06b
Title: Chemical tools for the interrogation of protein phosphorylation and pyrophosphorylation
Authors: Brown, Nathaniel
Advisors: Fiedler, Dorothea
Contributors: Chemistry Department
Keywords: Inositol pyrophosphates
Phosphopeptide enrichment
Phosphoproteomics
Phosphorimidazolides
Protein Pyrophosphorylation
Subjects: Chemistry
Biochemistry
Organic chemistry
Issue Date: 2018
Publisher: Princeton, NJ : Princeton University
Abstract: Protein phosphorylation is one of the most ubiquitous post-translational modifications (PTMs) in eukaryotes. Protein kinases catalyze the transfer of the γ-phosphoryl group of ATP onto an amino acid side chain, while phosphatases are responsible for phosphoryl group removal. Involved in almost every signaling pathway in the cell, the study of global phosphorylation, termed phosphoproteomics, has remained squarely in focus for several decades. Phosphoproteomics relies on the analysis of phosphoproteins digested into component peptides to monitor the phosphorylation state of the proteome. To improve identification, an enrichment step is required to separate phosphopeptides from non-phosphorylated peptides. Several well-characterized enrichment methods exist, but each has its limitations. Using phosphorimidazolide reagents, a class of molecules that react specifically with phosphoryl groups, a new chemical derivatization method for phosphopeptide enrichment was developed that is highly selective and efficient, and which aims to complement existing global phosphoproteomic enrichment methods. While phosphorylation is a crucial mode of signaling for many levels of cellular regulation, signaling by small-molecule second-messengers represents an equally important avenue for cells to respond rapidly to extracellular stimuli. One such class of signaling molecules, the inositol pyrophosphates, are a group of densely-phosphorylated messengers derived from the lipid-bound phosphatidylinositols. 5PP-InsP5, the most abundant mammalian inositol pyrophosphate isomer, has been linked to many key processes including insulin regulation, DNA damage repair, and obesity. Inositol pyrophosphates are thought to signal in part via a novel PTM termed pyrophosphorylation, in which the β-phosphoryl group of 5PP-InsP5 is transferred to a phosphoserine, forming a phosphoanhydride bond. To date, all evidence of protein pyrophosphorylation has been limited to in vitro observations that have relied on radioactive labeling, thus further tools are required to fully characterize the PP-InsPs and pyrophosphorylation. Towards this goal, an antibody-based approach was developed for the detection of pyrophosphorylation utilizing the enzymatically-synthesized thiophosphate analog 5PP-InsP5-βS. Additionally, a streamlined synthesis of 5PP-InsP5 was completed that allowed high quantities of pure 5PP-InsP5 to be produced and which provided a platform for efforts towards the chemical synthesis of 5PP-InsP5-βS in order to permit a more detailed examination of the breadth and depth of protein pyrophosphorylation.
URI: http://arks.princeton.edu/ark:/88435/dsp01qf85nf06b
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog: catalog.princeton.edu
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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