LEE, S.G.; BADER, B.D.; CHANG, E.S.; MYKLES, D.L.; Colorado State Univ., Fort Collins, CO; Colorado State Univ., Fort Collins, CO; Bodega Marine Lab, Bodega Bay, CA; Colorado State Univ., Fort Collins, CO: High-throughput proteomic approaches for identifying molt-associated proteins in crustacean molting gland

Regulation of the molting cycle in decapod crustaceans is mainly controlled by the X-organ/sinus gland (XO/SG) complex and Y-organ (YO). Many neuropeptide hormones are synthesized and secreted by eyestalk ganglia. One of these neuropeptide hormones, molt-inhibiting hormone (MIH), is considered the main regulator of ecdysteroidogenesis in YO. Eyestalk ablation (ESA) causes a rapid increase of ecdysteroid titers and induces molting. However, little information is known about the pathways for inhibition of ecdysteroidogenesis in YO after MIH triggers the signal cascade from its receptor on the YO membrane. Proteomics has become a powerful tool to identify proteins involved with specific physiological or disease states. In an effort to determine which proteins are involved in YO ecdysteroidogenesis, expression and cell-map proteomic approaches were used. Total protein extracts of YOs from intact and ES-ablated animals were analyzed by 2-D gel electrophoresis. ESA causes dramatic changes in the levels of proteins between 12 kDa and 27 kDa. These proteins were selected as putative candidates for molt-regulating factors from both silver and phosphoprotein-stained gels. Also, MIH interacting proteins were detected by immunoprecipitation with an anti-MIH antibody. These putative MIHBPs (MIH binding proteins) were observed only in intact YO, suggesting that these proteins are down-regulated in activated YOs. These proteins are being characterized by peptide mass fingerprinting and internal peptide sequencing using MALDI-TOF/TOF. Supported by NSF (IBN0-342982).