Equal amounts of ER proteins isolated from midguts of unfed and fed mosquitoes were separated by 12% SDS-PAGE and visualized using the GelCodeH Blue Staining Kit (Thermo Scientific, Rockford, IL, USA). Each gel lane was cut into 4 equal slices using a scalpel, and labeled as A, B, C, and D from high to low molecular weight regions. The gel slices were stored individually stored in a clean Eppendorf tube for in-gel digestion. Each gel slice was washed first with ddH 2 O for 15 min, then twice with 50% acetonitrile (ACN), and finally with 100 mM ammonium bicarbonate (Ambic, pH 8.0) containing 50% ACN for 15 min. After drying, the gel pieces were subjected to the standard in-gel digestion protocol. Briefly, proteins were reduced by 10 mM DTT/100 mM Ambic at 56uC for 45 min., and alkylated by 55 mM iodoacetamide (IAA)/ 100 mM Ambic at room temperature in the dark for 30 min. Gel pieces were washed with 100 mM Ambic dehydrated with ACN and dried. Proteolytic digestion was performed with 12.5 ng/ml trypsin dissolved in 100 mM Ambic and incubated on ice for 45 min. The digested mixture was acidified with 2% trifluoroacetic acid (TFA) in water for 1-2 minutes and then the supernatant was collected in a clean 1.5-ml tube. The peptides were extracted from the gel slice using 0.1% TFA in water, 0.1% TFA in 30% ACN, and 0.1% TFA in 60% ACN, respectively, with ultrasonication. The peptides extracted in the four steps were combined together, concentrated by a SpeedVac to a desired volume and subjected to LC-MS/MS analysis.