Proteome measurements for developing hypotheses about the biology of Drosophila.

摘要

Aging is one of the most challenging problems in biology. A fundamental understanding of this process at the molecular level does not exist. However, in-depth information about specific events associated with aging has recently begun to emerge. This growth is largely attributable to the fields of genomics and proteomics which provide high-throughput measurements of thousands of genes and proteins with each experiment. Aging research has also benefited from the use of model animal systems that provide greater feasibility in experimental design. The work presented below utilizes proteomics technology and the animal model, Drosophila melanogaster (commonly known as the fruit fly), in an attempt to elucidate the mechanisms of aging.;The work begins with the development of an analytical approach that couples reversed-phase liquid chromatography (LC) to ion mobility spectrometry (IMS) and mass spectrometry (MS) for the separation and detection of intact proteins. This was a novel approach that demonstrated the potential of LC-IMS-MS techniques for top-down proteomics analyses of simple protein mixtures. The bulk of the work, however, employs bottom-up shotgun proteomics techniques in which tryptic peptide mixtures are used as a means to identify the proteins present in whole-cell lysates. Semi-quantitative analyses are used to globally assess changes in the relative abundances of proteins in adult Drosophila across organism lifespan.;Various combinations of LC, strong-cation-exchange (SCX) chromatography, IMS, MS, and MS/MS methods are used to detect tryptic peptides from the heads or bodies of Drosophila. An initial survey of changes in wild type animals that revealed down regulation of metabolic-, reproduction- and development-related proteins, and changes in defense response is presented. These analyses provided clues about possible mechanisms of aging that led to biological experiments that examined the influence of prophenoloxidase, an immune response protein, on organism lifespan. The proteome was also measured with age under two conditions that increase Drosophila lifespan: ambient temperature and dietary restriction. Overall, this work provides evidence for many putative markers of aging at the protein level and insight to biological pathways associated with aging.