Submitted to: Journal of Biomolecular Techniques
Publication Type: Abstract Only
Publication Acceptance Date: March 23, 2010
Publication Date: September 1, 2010
Citation: Mclaughlin, M., Cilia, M., Howe, K.J., Gray, S.M., Thannhauser, T.W. 2010. Combined use of 2-D reverse phase chromatography & data independent mass spectrometry simultaneously characterizing proteomes of Schizaphis graminum & its obligate endosymbiont Buchnera aphidicola, from whole aphid extracts. Journal of Biomolecular Techniques. 21(3):S58. Technical Abstract: Understanding the molecular pathways coordinating protein biosynthesis and trafficking by aphids and the involvement of endosymbiont bacterium, Buchnera aphidicola in those processes, is critical to discovering the mechanisms of virus transmission by aphids as well as leveraging that knowledge towards development of a targeted approach for disrupting the spread of insect-vectored viruses. Yellow dwarf viruses are vectored by the green bug aphid, Schizaphis graminum. Yellow dwarf infections decimate the value of cereal crops by reducing seed yield. Here, we report the application of a novel reverse phase separation using the strong ion pairing agents, ammonium acetate or triethylammonium acetate in the first dimension coupled to an online, low pH reverse phase separation followed by data independent analysis, MSe, for the identification of proteins from the green bug. Proteomics studies in the green bug are challenging because there are little genomic resources available for database searching. However, genome of the related pea aphid, Acrythosiphon pisum is available and facilitated protein identification by homology-based database searching. Nearly 1000 proteins were identified from the green bug proteome with homologues in the pea aphid, including 190 hypothetical proteins and 80 proteins from the endosymbiont Buchnera proteomes. Several proteins previously associated with virus transmission were identified using this method, such as GroEL, actin, cuticle proteins, HSP70, GAPDH3, and even the low abundance proteins RACK-1, and cyclophilin, demonstrating the utility of this workflow for the discovery of proteins involved in virus transmission by aphids. With the exception of ubiquinated ribosomal proteins and cuticle proteins, both largely homologous across different taxa and genes, respectively, the 2-D reverse phase separation combined with the enhanced sampling and detection of peptides provided by MSe enabled us to assign specific protein identifications to 20% of the proteins identified, most of which were from the green bug proteome. Furthermore, the 2-D separation technique efficiently normalized the dynamic range of the sample, as we detected approximately 50 proteins from a pathogenic fungus of insects, previously uncharacterized in our green bug population.