Nanopore Identification of N-Acetylation by Hydroxylamine Deacetylation (NINAHD).

N -Acetyl modification, a chemical modification commonly found on biomacromolecules, plays a crucial role in the regulation of cell activities and is related to a variety of diseases. However, due to the instability of N -acetyl modification, accurate and rapid identification of N -acetyl modification with a low measurement cost is still technically challenging. Here, based on hydroxylamine deacetylation and nanopore single molecule chemistry, a universal sensing strategy for N -acetyl modification has been developed. Acetohydroxamic acid (AHA), which is produced by the hydroxylamine deacetylation reaction and serves as a reporter for N-acetylation identification, is specifically sensed by a phenylboronic acid (PBA)-modified Mycobacterium smegmatis porin A (MspA). With this strategy, N -acetyl modifications on RNA, DNA, proteins, and glycans were identified, demonstrating its generality. Specifically, histones can be treated with hydroxylamine deacetylation, from which the generated AHA can represent the amount of N -acetyl modification detected by a nanopore sensor. The unique event features of AHA also demonstrate the robustness of sensing against other interfering analytes in the environment.