Ryan Jack P, Slysz Gordon W, Rye Peter, Stow Sarah M, Dodds James N, Sausen John, Baker Erin S
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Agilent Technologies, Santa Clara, California 95051, United States.
J Am Soc Mass Spectrom. 2025 Jul 2;36(7):1493-1502. doi: 10.1021/jasms.5c00083. Epub 2025 Jun 9.
Synthetic oligonucleotides, such as antisense oligonucleotides or small interfering RNA, are small chain nucleic acid polymers that can be used therapeutically to control gene expression. Watson-Crick base pair interactions provide the primary mode of interaction between synthetic oligonucleotides and their target molecule, and this binding requires accurate, robust, and rapid sequence verification. The development of high-quality synthetic oligonucleotides and comprehensive analytical workflows for their evaluation is therefore essential. Herein, a platform coupling liquid chromatography, ion mobility spectrometry, collision-induced dissociation, and mass spectrometry (LC-IMS-CID-MS) was applied to facilitate oligonucleotide sequence confirmation. Using IMS, multiple charge states of the same oligonucleotide were mobility separated and analyzed simultaneously. Furthermore, all-ion fragmentation was implemented to provide sequence coverage for each charge state using fewer injections than current targeted multiple injection LC-MS methods. The results demonstrated herein denote sequence coverage is generally inversely proportional to oligonucleotide length (.., lower fidelity coverage for longer strands), with observed sequence coverages ranging between 40% and 80% for molecules comprised of 20-40 residues. To ease the burden of spectral interpretation and sequence determination for the LC-IMS-CID-MS data, an analysis workflow using the Pacific Northwest National Laboratories (PNNL) preprocessor and Agilent's BioConfirm software was developed.
合成寡核苷酸，如反义寡核苷酸或小干扰RNA，是小链核酸聚合物，可用于治疗性控制基因表达。沃森-克里克碱基对相互作用是合成寡核苷酸与其靶分子之间的主要相互作用方式，这种结合需要准确、可靠且快速的序列验证。因此，高质量合成寡核苷酸的开发及其评估的综合分析工作流程至关重要。在此，应用了一种结合液相色谱、离子淌度质谱、碰撞诱导解离和质谱（LC-IMS-CID-MS）的平台来促进寡核苷酸序列确认。使用离子淌度质谱，同一寡核苷酸的多个电荷态被进行淌度分离并同时分析。此外，实施全离子碎裂以使用比当前靶向多次进样液相色谱-质谱方法更少的进样次数为每个电荷态提供序列覆盖。本文所示结果表明序列覆盖通常与寡核苷酸长度成反比（即，较长链的保真度覆盖较低），对于由20至40个残基组成的分子，观察到的序列覆盖范围在40%至80%之间。为减轻LC-IMS-CID-MS数据的谱图解读和序列确定负担，开发了一种使用太平洋西北国家实验室（PNNL）预处理器和安捷伦BioConfirm软件的分析工作流程。
