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Updated: Aug 21, 2026

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications
Published on: July 10, 2020
Rapid Exonuclease-Based De Novo Sequencing Strategy for Long Oligonucleotides via Liquid-Phase Ion Trap Coupled With
Song Lu1, Jie Hong2, Xinyan Fu3
1Center for Advanced Measurement Science, National Institute of Metrology, Beijing, China.
Abstract:
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.
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