UVM Theses and Dissertations
Format:
Print
Author:
Henkin, Joshua Adam
Dept./Program:
Cell and Molecular Biology Program
Year:
2004
Degree:
Ph. D.
Abstract:
Proteomics, the wide scale characterization of protein products from a genome, is a new field of study that describes the qualitative and quantitative differences between two states of a cell, tissue or organism. One of the major challenges in the field of proteomics is to identify proteins and examine their binding interactions. The overall goal of this dissertation was to characterize the Drosophila indirect flight muscle (IFM) proteomes in wild-type and mutant strains through one and two dimensional gel electrophoresis, mass spectrometry and muscle mechanics. Drosophila IFM is an excellent model for this type of research because specific proteins from the IFM can be altered by a mutation while preserving the viability of the fly. The effect of these mutations can be studied in vivo and characterized to identify putative protein-protein interactions (PPI).
Chapter I reviews traditional and modern methods used to identify PPI with an emphasis on techniques that permit the identification of PPI via high throughput technology. The concept of mutational proteomics is introduced as an approach for utilizing Drosophila IFM as a model for examining functional PPI by comparing proteomes of different IFM mutants to monitor how a mutation affects the expression of other proteins in the proteome. Proteome analysis was accomplished by gel separation followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for protein identification. Chapter 2 describes a simple method enhancement that reduces the number of non sample masses submitted to database search engines used to identify proteins. This technique improved the significance of 6 out of 10 control sample identifications as well as 7 out of 16 Drosophila IFM putative protein identifications.
The method described in Chapter 2 was used extensively in Chapter 3, which details a proteome wide analysis of IFM myofibrillar proteins from wild-type and two mutant Drosophila strains, actin null (Act88FKM⁸⁸) and myosin null (Mhc⁷). In the wild-type myofibrillar proteome, 22 of the 28 detected proteins were identified and compared to proteins expressed in mutant proteomes to determine the effect of those specific mutations on the proteome. This data was used to test the currently accepted theory that mutations in thick filaments do not affect thin filament assembly and vice versa. The results indicated that the mutations might exert a wider range of changes in the proteome than previously expected. Results from this study also revealed many putative PPI in need of further analysis and confirmation via direct binding assays.
Chapter I reviews traditional and modern methods used to identify PPI with an emphasis on techniques that permit the identification of PPI via high throughput technology. The concept of mutational proteomics is introduced as an approach for utilizing Drosophila IFM as a model for examining functional PPI by comparing proteomes of different IFM mutants to monitor how a mutation affects the expression of other proteins in the proteome. Proteome analysis was accomplished by gel separation followed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry for protein identification. Chapter 2 describes a simple method enhancement that reduces the number of non sample masses submitted to database search engines used to identify proteins. This technique improved the significance of 6 out of 10 control sample identifications as well as 7 out of 16 Drosophila IFM putative protein identifications.
The method described in Chapter 2 was used extensively in Chapter 3, which details a proteome wide analysis of IFM myofibrillar proteins from wild-type and two mutant Drosophila strains, actin null (Act88FKM⁸⁸) and myosin null (Mhc⁷). In the wild-type myofibrillar proteome, 22 of the 28 detected proteins were identified and compared to proteins expressed in mutant proteomes to determine the effect of those specific mutations on the proteome. This data was used to test the currently accepted theory that mutations in thick filaments do not affect thin filament assembly and vice versa. The results indicated that the mutations might exert a wider range of changes in the proteome than previously expected. Results from this study also revealed many putative PPI in need of further analysis and confirmation via direct binding assays.