Proteome-Wide Drug Target Identification and Binding Site Mapping Using LiP-MS

Target identification in a physiologically relevant context is a critical step in both target-based and phenotypic drug discovery. Limited proteolysis coupled with mass spectrometry (LiP-MS) has recently emerged as a robust strategy for deconvoluting the protein targets of small molecules and peptides directly in complex cell lysates, without requiring chemical derivatization of compounds or genetic engineering of cell models. In LiP-MS, a broadly active protease is applied under controlled conditions, generating peptide fingerprints that reflect drug-induced structural perturbations or steric protection at the protein level. When combined with quantitative mass spectrometry, this approach can interrogate more than 250,000 peptides, corresponding to up to ~9,000 proteins in the proteome.

In addition to global target identification, we developed a high-resolution LiP (HR-LiP) workflow for targeted characterization of small molecule–protein interactions directly in cell lysates from overexpression systems. This approach eliminates the need for protein purification, avoiding artifacts such as truncation or misfolding.