High-ratiO partiaL proteolysiS with carriER proteome (HOLSER) Enables Global Structure Profiling and Site-resolved Elucidation of Ligand–Protein Interactions

Understanding how cellular proteins interact with their environment, including endogenous and exogeneous molecules, is critical for elucidating mechanisms of cellular regulation and drug action. Proteolysis-based stability profiling methods such as peptide-centric local stability assay (PELSA) offer peptide-level resolution of ligand-induced conformational changes but are limited by modest proteome coverage and depth, as well as high variability. To overcome these limitations, we developed high ratio partial proteolysis with carrier proteome (HOLSER), an efficient workflow that combines high enzyme-to-substrate ratio with extended digestion time for low peptide yield variability and tandem mass tag (TMT)-based multiplexing that includes full digests for enhanced proteome depth and sequence coverage as well as precision of abundance measurements. We demonstrate HOLSER’s capabilities across diverse usage cases, including global proteome structure profiling, proof-of-principle methotrexate target elucidation, kinase target mapping with staurosporine, and high-resolution analysis of the FKBP–mTOR complex response to rapamycin. HOLSER reveals domain-specific stabilization and identifies peptides in direct contact with ligands, providing a powerful and scalable platform for simultaneous probing structural changes at the scale of the whole proteome, individual protein domains and specific binding sites.