Insect Protein Extraction and Identification

Half of the last four segments of each insect abdomen were isolated using a razor blade and scissors which were cleaned after each sample with a 50% bleach solution. The insect tissue was mixed with 800 to 1200 μL of denaturing buffer (5% blue bromophenol, 150 mM Tris pH 6.8, 2% SDS, 5% β-mercaptoethanol, 7.8% glycerol) depending on the amount of tissue, vortexed for 20 seconds, and heated at 95° C for five minutes. The proteins were then separated via SDS-PAGE and stained with Coomassie blue. Hemoglobin (approximately 16 kDa) was excised from the gel for trypsin digestion, as previously described [13 (link)] The LC-MS/MS analysis was carried out as previously described [13 (link)], and employed a LTQ (linear trap quadrupole)-Orbitrap Discovery with a Finnigan Surveyor Pump Plus and Micro AS autosampler (Thermo Electron; San Jose, CA, USA) controlled with Xcalibur™ 2.1 Software (Thermo Fisher Scientific, Inc.; Waltham, MA, USA). Precursor ion spectra were obtained in the orbitrap, and fragment ion spectra were obtained in the linear ion trap.