Doctoral Dissertations

Date of Award

12-2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Life Sciences

Major Professor

Robert L. Hettich

Committee Members

Alison Buchan, Loren J. Hauser, Cynthia B. Peterson, Brynn H. Voy

Abstract

Proteomics comprises the identification and characterization of the complete suite of expressed proteins in a given cell, organism or community. The coupling of high performance liquid chromatography (LC) with high throughput mass spectrometry (MS) has provided the foundation for current proteomic progression. The transition from proteomic analysis of a single cultivated microbe to that of natural microbial assemblages has required significant advancement in technology and has provided greater biological understanding of microbial community diversity and function.

To enhance the capabilities of a mass spectrometric based proteomic analysis, an integrated approach combining bioinformatics with analytical preparations and experimental data collection was developed and applied. This has resulted in a deep characterization of the extracellular fraction of a community of microbes thriving in an acid mine drainage system. Among the notable features of this relatively low complexity community, they exist in a solution that is highly acidic (pH < 1) and hot (temperature > 40°C), with molar concentrations of metals. The extracellular fraction is of particular interest due to the potential to identify and characterize novel proteins that are critical for survival and interactions with the harsh environment.

The following analyses have resulted in the specific identification and characterization of novel extracellular proteins. In order to more accurately identify which proteins are present in the extracellular space, a combined computational prediction and experimental identification of the extracellular fraction was performed. Among the hundreds of proteins identified, a highly abundant novel cytochrome was targeted and ultimately characterized through high performance MS. In order to achieve deep proteomic coverage of the extracellular fraction, a metal affinity based protein enrichment utilizing seven different metals was developed and employed resulting in novel protein identifications. A combined top down and bottom up analysis resulted in the characterization of the intact molecular forms of extracellular proteins, including the identification of post-translational modifications. Finally, in order to determine the effectiveness of current MS methodologies, a software package was designed to characterize the > 100,000 mass spectra collected during an MS

experiment, revealing that specific optimizations in the LC, MS and protein sequence database have a significant impact on proteomic depth.

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