Drug Target ID and Binding Site Mapping in Complex Cellular Environments Using LiP-MS

Target identification in a cellular context is a crucial step in both target-based and phenotypic drug discovery. Limited proteolysis coupled with mass spectrometry (LiP-MS) has emerged as a powerful technique for target deconvolution of small molecules and peptides in cell lysates, eliminating the need for compound modifications or genetic manipulation of cell lines. LiP-MS employs a non-specific protease under well-controlled conditions to detect drug-induced structural changes or steric hindrance on protein targets. Using quantitative mass spectrometry, this method can analyze over 250,000 peptides, covering up to 9,000 proteins in the proteome (Fig. 1). To enhance target identification, the workflow integrates a seven-concentration dose-response experiment alongside a machine-learning framework, which calculates a LiP-score to rank potential targets and predict possible binding sites (Fig. 2).

Here, we demonstrate the performance of the LiP-MS approach on the TrueTarget platform using three compounds: two kinase inhibitors and a molecular glue degrader.