Post-translational modifications of SEL24K from salmon eggs and ZPA from Xenopus laevis eggs

<p>Post-translational modifications (PTMs) of proteins play significant roles in regulation of biological activities and signal transduction. Examining their diversity is critical for understanding the mechanisms of cellular regulations. Among the various techniques employed for identification of PTMs, mass spectrometry has become a more and more important tool for detecting and mapping these covalent modifications and quantifying their changes. The two projects described in this dissertation focus mainly on the method development for characterization of two major PTMs, disulfide bonds and glycosylation. In the first project, the disulfide bond pattern of a rhamnose-binding lectin SEL24K from the Chinook salmon Oncorhynchus tshawytscha was assigned unambiguously based on a multi-enzyme digestion strategy in combination with MALDI-TOF mass spectrometry analysis. The disulfide bond pattern was found to be symmetrical in the tandem repeat sequence of SEL24K. More importantly, an interesting phenomenon of gas-phase scrambling of disulfide bonds was observed during MALDI mass spectrometry analysis and a possible mechanism for this surprising scrambling was proposed. To the best of our knowledge, this is the first report of disulfide bond scrambling in the gas phase during MALDI-MS analysis. This observation has important ramifications for unambiguous assignment of disulfide bonds. In the second project, the glycosylation of a glycoprotein ZPA from the vitelline envelope of Xenopus laevis was determined by applying a strategy of general proteolysis coupled with mass spectrometry. The vitelline envelope glycoproteins were first separated through SDS-PAGE. A nonspecific in-gel pronase digestion was performed on the excised band of ZPA to produce informative small glycopeptides. Lectin affinity chromatography was used for the enrichment of these glycopeptides. An in-gel PNGase F digestion was also carried out to release the N-linked glycans from ZPA. The enriched glycopeptides and glycans were finally analyzed by MS and MS/MS techniques on MALDI-TOF and MALDI-TOF/TOF instruments.</p>

Name thesis:degree_name

Doctor of Philosophy (Ph.D.)

Level thesis:degree_level

Dissertation - Pacific Access Restricted

Discipline thesis:degree_discipline

Pharmaceutical and Chemical Sciences

Year dc:date.available

2011

Author dc:creator

• Zhao, Liang

Contributors dc:contributor

• Andreas Franz

• Analytical chemistry
• Biochemistry
• Developmental biology
• Health and environmental sciences
• Pure sciences
• Disulfide bonds
• Glycosylation
• Posttranslational modifications
• Chemicals and Drugs
• Chemistry
• Medical Pharmacology
• Medicinal-Pharmaceutical Chemistry
• Medicine and Health Sciences
• Pharmaceutical Preparations
• Pharmacy and Pharmaceutical Sciences
• Physical Sciences and Mathematics

Licence

• http://rightsstatements.org/vocab/InC/1.0/

Identifier

9781124933757

OAI identifier oai:identifier

oai:scholarlycommons.pacific.edu:uop_etds-1159