Nanopore‐Based High Resolution Detection of Multiple Post‐Translational Modifications in Proteins

Protein post‐translational modifications (PTMs) play crucial roles in various cellular processes. Despite their significance, only a few PTMs have been extensively studied at the proteome level, primarily due to the scarcity of reliable, convenient, and low‐cost sensing methods. Here, we present a straightforward and effective strategy for detecting PTMs on short peptides through host‐guest interaction‐assisted nanopore sensing. Our results demonstrate that the identity of 13 types of PTMs in a specific position of a phenylalanine‐containing peptide could be determined via current blockage during translocation of the peptide through α‐hemolysin nanopores in the presence of cucurbit[7]uril. Furthermore, we extend this strategy by incorporating a short peptide into the probe, enabling the discrimination of various PTMs, positional isomers, and even multiple PTMs on the target peptide. With ongoing improvements, our method holds promise for practical applications in sensing PTMs in biologically relevant samples, offering an efficient alternative to traditional mass spectrometry approaches.