On-bead tryptic proteolysis: an attractive procedure for LC-MS/MS analysis of the Drosophila caspase 8 protein...

This study aims to characterize the immune response against bacteria in Drosophila melanogaster. Obtaining a description of the in vivo state of protein complexes requires their isolation as a snapshot of physiological conditions before their identification. Affinity purification with streptavidin-biotin system is widely used to address this issue. However, because of the extraordinary stability of the interaction between streptavidin and biotin, the release of biotin-labeled bait remains a challenge. We transfected Drosophila cells with a DNA construct encoding a biotin-tagged Dredd protein (ortholog of caspase 8). After affinity purification, different strategies were evaluated, and proteins analyzed by LC-MS/MS mass spectrometry. The on-bead digestion allowed the identification of more proteins associated to the Dredd complex than different protocols using competitive or acid elution. A functional assay showed that a large part of the proteins specifically identified in the Dredd sample are functionally involved in the activation of the Imd pathway. These proteins are immune response proteins (BG4, Q9VP57), stress response proteins (HSP7C, Q9VXQ5), structural proteins (TBB1, CP190), a protein biosynthesis protein (Q9W1B9) and an antioxidant system protein (SODC). Our results clearly show that on-bead digestion of proteins is an attractive procedure for the study of protein complexes by mass spectrometry. This article is part of a Special Issue entitled: Translational Proteomics.