Date of Award

2010

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Keywords

Pure sciences, Cellular stress, Disulfide isomerase, Protein modifications

Supervisor

Vacratsis, Otis

Rights

info:eu-repo/semantics/openAccess

Abstract

In recent years mass spectrometry has become an invaluable tool to address an array of biological questions. The versatility of this experimental approach does not only allow assignment of protein identity and identification of sequence specific modifications, but with the help of particular derivatization techniques facilitates the determination of peptide quantity. Each of these approaches were applied to the following biological projects:

The 21 kDa heat stable protein purified from the encysted embryo of Artemia franciscana was characterized by time-of-flight mass spectrometry. De novo sequencing of peptides identified this protein as a group 1 Late embryogenesis abundant (LEA) protein. The amino acid sequence assignment to these peptides allowed amplification of the entire gene sequence from an embryonic cDNA library. This was deposited into the NCBI database (EF656614). The expression of group 1 LEA protein is consistent with and supports a role in desiccation tolerance. In addition, this is a first report describing identification of a group 1 LEA protein in an animal species.

A MS-based quantitative analysis was performed in order to analyze relative changes in the dynamic thiol and disulfide states of the redox sensitive protein disulfide isomerase, PDI. PDI cysteine residues were derivatized with an isotope-coded affinity tag (ICAT), thus allowing a direct comparison between the reduced and auto-oxidized forms. Quantitation was based on relative ratios between light and heavy isotopically labeled cysteine containing peptides. The application of the ICAT-labeling approach to PDI related studies, allowed direct assignment of individual cysteine residues and their oxidation status, compared to the previously employed techniques, that only provided information regarding the average number of modified cysteine residues within PDI, not their exact identity.

A combination of a phosphopeptide enrichment step and a MS-based approach was utilized to identify three phosphorylation sites on hYVH1, an atypical dual specificity phosphatase that functions as a cell survival factor. With the help of phosphomimetic and non-phosphorylable mutants, we were able to decipher their effect on localization and progression through the cell cycle.

Collectively, these studies manifest the power of MS-generated data to influence and guide many different fields ranging from molecular embryology to biochemistry.

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