To more directly identify midgut proteins that might be contributing to whorl formation in unfed adult female mosquitoes, we isolated microsomal proteins from midgut tissues of unfed and amino acid fed mosquitoes (30 min post-feeding) using a modified procedure that minimized protease activity in the sample and enriched for RER associated proteins (see Materials and Methods). The protein samples were separated by molecular weight using SDS PAGE and four gel slices from each lane were processed for LC-MS/MS analysis and protein annotation ( Figure 4 ). The SDS PAGE analysis revealed that intact proteins of up to ,130 kDa were present in both samples, suggesting that protease activity was minimal in the midgut protein sample from fed mosquitoes. Moreover, no major differences were observed in the distribution of proteins isolated from the unfed or amino acid fed mosquitoes, indicating that the 30 min time point preceded feeding-induced protein synthesis.