Short-gradient LC-MS and automated sample preparation for LiP-MS

Drug development faces a significant bottleneck due to the high failure rate of candidate drugs during clinical translation. Efficiently identifying drug targets and potential off-targets is crucial to address this challenge. Technologies such as limited proteolysis coupled with mass spectrometry (LiP-MS) aim to overcome this hurdle. LiP-MS (Figure 1) offers a unique approach for unbiased drug target deconvolution and binding site identification in complex proteomes. By capturing the conformational changes or steric hinderance that occur when a drug binds to its target protein, LiP-MS enables the differentiation of true drug targets from non-targets and off-targets based on their distinct structural responses. However, current LiP-MS protocols remain challenging, low in throughput, and manual labor-intensive, which restricts their use in larger phenotypic screening for drug discovery. Here, we present the successful transfer of our current manual LiP-MS pipeline (Piazza et al. 2020) to a fully automated sample preparation workflow in combination with short gradient LC-MS methods.