Author's School

Graduate School of Arts & Sciences

Author's Department/Program

Chemistry

Language

English (en)

Date of Award

January 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Chair and Committee

Richard Gross

Abstract

The reversible covalent phosphorylation of cellular proteins is widely believed to be the most important mechanism for the regulation of multiple signal transduction pathways in cell growth, division, and death by acting as a molecular switch at multiple nodes in metabolic networks. In this dissertation, first, a novel mass spectrometric strategy is reported that exploited the unique chemical properties of thiocholine that was introduced into protein phosphosites through alkaline beta-elimination and Michael addition: BEMA), allowing the specific detection, identification and quantitation of phosphorylated serine/threonine containing peptides. Through replacement of the phosphate with thiocholine as the Michael donor, this strategy resulted in a marked increase in ionization sensitivity during ESI accompanied by enhanced peptide sequence coverage during CID. Moreover, the definitive localization of phosphorylated residues is greatly facilitated through the generation of diagnostic triads of fragment ions resulting from peptide bond cleavage and further neutral loss of either trimethylamine: -59 Da) or thiocholine thiolate: -119 Da) during CID in tandem mass spectrometric analyses such as MS2 and MS3. Synthesis of stable isotope labeled thiocholine enabled the quantitation of protein phosphorylation with high precision by ratiometric comparisons. The utility of this approach was demonstrated in an intact cell system through identification of the endogenous phosphorylation sites in iPLA2β; during heterologous expression in Sf-9 cells. A total of 12 unique phosphopeptides and 19 phosphorylation sites were identified with the developed strategy whereas the conventional approach identified only five peptides and six phosphorylation sites. Lastly, the BEMA strategy was applied to in vivo tissue system for the quantitative analysis of the murine myocardial mitochondrial phosphoproteome following cardiac ischemia. A total of 36 phosphopeptides from 35 mitochondrial proteins with 50 phosphosites: 37 of which were previously unknown), were identified. Collectively, we have demonstrated beta-elimination of phosphate and subsequent Michael addition: BEMA) with natural abundance and stable isotope labeled thiocholine is an effective strategy for in vivo quantitative phosphoproteomics of both cell-based and tissue-based systems.

DOI

https://doi.org/10.7936/K7G15XWN

Comments

Permanent URL: http://dx.doi.org/10.7936/K7G15XWN

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