Rapid Exonuclease‐Based De Novo Sequencing Strategy for Long Oligonucleotides via Liquid‐Phase Ion Trap Coupled With Mass Spectrometry

The sequencing of synthetic oligonucleotides is fundamental to life sciences and therapeutics, yet current analytical approaches present inherent technical drawbacks of varying degrees. Hybridization-based techniques cannot sequence the oligonucleotide primers they rely on, while the accuracy of mass spectrometry (MS)-based techniques significantly diminishes as fragment length increases. Here, we introduce a liquid-phase ion trap (LPIT) coupled with electrospray ionization (ESI)-MS as a solution for rapid, high-fidelity oligonucleotide sequencing. The LPIT utilizes a strong electric field to efficiently dissociate confounding metal cation adducts prior to MS analysis, resulting in cleaner spectra and enhanced signal intensity. This "desalting-first" approach enables direct, rapid oligonucleotide analysis, achieving mass measurement accuracy comparable to benchmark liquid chromatography-ESI-MS (LC-ESI-MS) platforms. By integrating this platform with an exonuclease digestion workflow, we achieved full sequence coverage of oligonucleotides in under 30 min, a dramatic improvement in speed over existing methods. This work establishes LPIT-ESI-MS as a powerful platform for the direct and rapid sequencing of long oligonucleotides, addressing a critical unmet need in biotechnology and drug development.