SP3-PELSA: A rapid and sensitive approach for studying protein-ligand interaction with small-amount samples

Deciphering protein-ligand binding events is essential for unraveling biological pathways and advancing drug development. The recently developed peptide-centric local stability assay (PELSA) has demonstrated superior performance compared to other mainstream approaches. However, the conventional PELSA approach relies on filter-based peptide recovery, which results in prolonged processing time and relatively low yields, particularly when operating with limited starting material. To address these limitations, we present an optimized PELSA approach termed SP3-PELSA, which leverages the simple and efficient peptide recovery of the Single-Pot, Solid-Phase-enhanced Sample Preparation (SP3) method. Through a systematic evaluation of protein precipitation and trypsin inactivation conditions, we identified 1% formic acid and 80% acetonitrile as the optimal condition. A comparative analysis of SP3-PELSA with conventional PELSA revealed that SP3-PELSA reduces the processing time by fivefold and significantly increases the peptide recovery rate, especially for hydrophobic peptides. Consequently, SP3-PELSA outperformed other mainstream approaches in the identification of established drug targets. Owing to its high recovery efficiency and potential for automation, SP3-PELSA holds great promise for large-scale drug target discovery, especially in the context of scarce clinical samples. Furthermore, its exceptional recovery of hydrophobic peptides positions this approach as a powerful tool for analyzing highly hydrophobic proteins in drug target screening.