Dissecting Cell Signalling Network Rewiring with Synthetic Antibodies

To understand the molecular basis of cellular functions, we are aiming at characterizing endogenous molecular complexes directly purified from biological samples. Hybridoma derived antibodies are the reagent of choice to purify endogenous protein complexes before they can be analyzed by mass spectrometry. However this strategy is hampered by the time and expense of generating antibodies of high affinity and specificity. In contrast phage display affinity reagents can provide a strategy to systematically develop affinity reagents for application to a variety of targets (PI: 18488004). To examine the efficacy of applying affinity reagents to systematic proteomics, we chose to investigate the use of synthetic antibody fragments (Fabs) for mass spectrometry characterization of protein interaction networks (interactomes), by (i) developing Fab-based methods to efficiently purify intact protein complexes, (ii) benchmark Fab-based affinity precipitation / mass spectrometry (AP/MS) against Flag-based established AP/MS methods, (iii) assess the performance of Fab-based AP/MS to characterize endogenous protein complexes in different signalling states.