Precise Identification of Native Peptides with Posttranslational Proline Hydroxylation by Nanopore

Hydroxylation, an extensive post‐translational modification on proline, is critical for the modulation of protein structures, further dominating their functions in life systems. However, current mass spectrometry‐based identification, could hardly distinguish hydroxylation from neighboring oxidation due to the same mass shifts, as well as challenges posed by low abundance and exogenous oxidation during sample preparation. To address these, an engineered nanopore was designed, capable of discriminating single hydroxyl group, to achieve the identification of proline hydroxylation on individual native peptides directly in the mixture. By modeling the interactions between hydroxylated proline and its specific recognition protein, we introduced a hydrophobic region in aerolysin lumen with A224Y/T274W mutations to enhance the sensitivity for proline residue. The results showed that proline hydroxylation on native HIF‐1α fragments could be unambiguously identified without purification, which could be maintained even in the presence of neighboring oxidation. The voltage‐dependent experiments further demonstrated more relaxed peptide structures induced by hydroxylation, supporting the great impact of hydroxylation on chemical properties of proline and the molecular mechanism of the specific recognition for hydroxylated peptides in nature. These findings highlight the potential of nanopore for precise hydroxylation detection, offering a reliable platform for further uncovering the related functions in biological systems.