Studying Protein-Ligand Interactions by Protein Denaturation and Quantitative Cross-Linking Mass Spectrometry

Recently, several mass spectrometry methods have utilizedproteinstructural stability for the quantitative study of protein-ligandengagement. These protein-denaturation approaches, which include thermalproteome profiling (TPP) and stability of proteins from rates of oxidation(SPROX), evaluate ligand-induced denaturation susceptibility changeswith a MS-based readout. The different techniques of bottom-up protein-denaturationmethods each have their own advantages and challenges. Here, we reportthe combination of protein-denaturation principles with quantitativecross-linking mass spectrometry using isobaric quantitative proteininteraction reporter technologies. This method enables the evaluationof ligand-induced protein engagement through analysis of cross-linkrelative ratios across chemical denaturation. As a proof of concept,we found ligand-stabilized cross-linked lysine pairs in well-studiedbovine serum albumin and ligand bilirubin. These links map to theknown binding sites Sudlow Site I and subdomain IB. We propose thatprotein denaturation and qXL-MS can be combined with similar peptide-levelquantification approaches, like SPROX, to increase the coverage informationprofiled for facilitating protein-ligand engagement efforts.