Proteomic Analysis of Individual Drosophila Hemolymph
thesisposted on 2017-03-10, 00:00 authored by Qi Zeng
Developing analysis methods and tools to understand the chemical information of the proteome of an organism of limited volume are challenging and vital for modern proteomic studies. Drosophila melanogaster (also called fruit fly) is one of the ideal animal models for the study of proteomic composition because of the high degree of genome homology with the human genome. Nevertheless, it has less than 50 nL blood (hemolymph) available for collection due to its small size (about 3 mm). The goal of this thesis is to present sample preparation and analysis methods to improve the identification of proteins in volume-limited biological samples using a series of chromatography and mass spectrometry methods. A hyphenated nano-reverse phase liquid chromatography chip column-mass spectrometry (nano-RPLC chip column-MS) method was developed to obtain proteomic information of hemolymph from an individual fruit fly. This is the first study to report a qualitative analysis of proteomic composition of hemolymph from individual adult female fruit flies. A microliter-scale protein digestion protocol was also developed to assist the digestion efficiency of limited volume sample. With the improved sample preparation method, six novel proteins were identified for the first time at the translation level. Detection of 13 proteins that are well-known in the literature speaks to the method’s validity and demonstrates the ability to reproducibly analyze volume-limited samples from individual fruit flies for protein content. Further, comprehensive prefractionation methods were developed by fabricating 2-cm long chromatography columns for individual fruit fly hemolymph samples. Detection of lower abundance proteins was enhanced with reverse phase and ion exchange prefractionation methods when followed by high mass resolution and high mass accuracy fourier transform ion cyclotron resonance-mass spectrometry (FT-ICR) and Orbitrap mass spectrometers. CE was brought on-line connected with a portable ion trap mass spectrometer by developing an interface. The sheathless CE-MS hyphenated instrument, built in house, enables efficient separation and detection of a list of amino acid standards and provides an alternative fast separation and detection method for small molecule analysis. This coupled CE-MS instrument compliments the complicated FT-ICR tandem mass spectrometry identification for larger molecular weight protein samples.