Qualitative and Quantitative Proteomics Methods for the Analysis of the Anopheles gambiae Mosquito Proteome

Abstract

Anopheles gambiae is the major African malaria vector. Insecticide and drug resistance highlight the need for novel malaria control strategies. A. gambiae exhibits daily (diel and/or circadian) rhythms in physiology and behavior that include flight, mating, sugar and blood-meal feeding, and oviposition. Olfaction is important for detecting blood-feeding hosts, sugar feeding sources, and oviposition sites. We previously reported on mRNA array-based changes in gene expression under light-dark cycle (diel) and constant dark (circadian) conditions. We were able to characterize 25 known or putative olfactory genes in female heads. We sought to follow up on these reported changes in gene expression and correlate them with expected changes in protein response. Here, we describe our recently developed methods and meta-level results for both qualitative and differential proteomics analyses of A. gambiae mosquitoes collected in a time-of-day-specific framework to assess temporal changes in protein abundance over the 24-h day. The traditional challenges associated with proteomics are amplified in an insect such as the mosquito, which contains a large amount of non-proteinaceous material associated with the cuticle and trachea and a high dynamic background of proteins associated with flight muscles and oxidative metabolism (e.g., myosin, glutathione S-transferases). We thus sought to use targeted, quantitative proteomics to directly measure differences in protein abundance in a time-of-day-dependent manner. We used multiple reaction monitoring (MRM), which has the advantage of being able to probe selected target lists with high sensitivity, wide dynamic range, and good/excellent reproducibility. We first characterized proteins in a qualitative format and subsequently examined subsets of specific proteins of interest in a high-fidelity quantifiable approach. Targeted quantitative multiple/single reaction monitoring (MRM/SRM) proteomics allowed for the measurement of changes in protein abundance in a time-of-day-specific manner over the 24-h diel cycle. Utilizing this accurate technique requires robust and reproducible protein/peptide preparation techniques in order to obtain consistent data. Here, we describe a technique using liquid N-2 homogenization-based protein extraction and proteolytic digestion applied to multiple discrete tissues (whole heads, antennae, total head appendages, compound eyes, and bodies), with subsequent liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based analysis of the resulting tryptic peptides. This technique is largely portable and should function well in any arthropod system with little modification. The results of our analyses are the generation of tissue-discrete determination of peptides, targeted quantitative analysis of peptides, and the deposition of datasets in VectorBase.org for use by the vector biology, arthropod, and proteomics research communities.