Towards chromatographic and proteomic analysis of protein complexes

Qiang Gao, Purdue University

Abstract

The study of intermolecular assemblies of proteins is a new frontier in protein chemistry and biology. Proteins often assemble into spatially coordinated complexes of distinct structure, composition, and function. At least 33% of the proteins in yeast are part of one or more protein complex at some time. Concerted efforts have been made during the past decade to study the structure, composition, aggregation, dissociation, and function of specific protein complexes along with attempts to understand how external stimuli impact these processes. Even with the expenditure of so much effort, methods for studying the cellular interactome are still not comparable in speed and universality to those used in proteomics and genomics. In this thesis, Size Exclusion Chromatography (SEC) was incorporated into global proteomics methods in such a way that proteins in complexes could be recognized, identified, and characterized in response to stimulus from the cellular medium. In the first part of this work, different kinetic conditions such as salt, PH and storage temperature on the stabilities of protein complexes were evaluated. Protein complexes eluted from different fractions of SEC chromatography were identified in optimized conditions, and particular attention was focused on recognizing HUB proteins and their interaction partners. In the second part of this work, by utilizing biotin hydrazide labeling integrated with avidin affinity chromatography, we successfully characterized the oxidation sites on the HUB protein complexes induced by acetic acid treatment. We found that the oxidation sites on distinct complexes are different from each other, which reveals that the binding partners accommodate the tertiary structures of the HUB proteins differently and oxidized parts on the HUB proteins were greatly determined by their accessibility to reactive oxygen species (ROS). Interestingly, the oxidation sites were either located on or adjacent to the functional residues of the proteins. This would possibly explain the mechanism of oxidative damage on the enzyme activity of protein complexes. In the third part of this work, SEC was integrated with the quantification methods involving stable isotope labeling by incorporation of amino acids in cell culture (SILAC) to characterize the dynamics of the protein complexes under oxidative stress induced by acetic acid treatment. Our method has demonstrated the capability in studying the dynamics of protein complexes from energy metabolism pathway in the yeast cells. Furthermore, we discovered that the Enolase II (ENO2) was covalently modified by a small Ubiquitin like protein of the SUMO family (SMT3), which might explain the unknown mechanism of ENO2 being transported to different cellular compartments to perform anti-disease functions.

Degree

Ph.D.

Advisors

Regnier, Purdue University.

Subject Area

Analytical chemistry|Biochemistry

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