Department of Chemistry, University of Texas at Austin, Austin, Texas 78712, United States.
Protein Metrics Inc., 20863 Stevens Creek Boulevard, Cupertino, California 95014, United States.
J Am Soc Mass Spectrom. 2022 Mar 2;33(3):510-520. doi: 10.1021/jasms.1c00340. Epub 2022 Feb 14.
With the increased development of new RNA-based therapeutics, the need for robust analytical methods for confirming sequences and mapping modifications has accelerated. Characterizing modified ribonucleic acids using mass spectrometry is challenging because diagnostic fragmentation may be suppressed for modified nucleotides, thus hampering complete sequence coverage and the confident localization of modifications. Ultraviolet photodissociation (UVPD) has shown great potential for the characterization of nucleic acids due to extensive backbone fragmentation. Activated electron photodetachment dissociation (a-EPD) has also been used as an alternative to capitalize on the dominant charge-reduction pathway prevalent in UVPD, facilitate dissociation, and produce high abundances of fragment ions. Here, we compare higher-energy collisional activation (HCD), UVPD using 193 and 213 nm photons, and a-EPD for the top-down sequencing of modified nucleic acids, including methylated, phosphorothioate, and locked nucleic acid-modified DNA. The presence of these modifications alters the fragmentation pathways observed upon UVPD and a-EPD, and extensive backbone cleavage is observed that results in the production of fragment ions that retain the modifications and allow them to be pinpointed. LNA and 2'--methoxy phosphorothioate modifications caused a significant suppression of fragmentation for UVPD but not for a-EPD, whereas phosphorothioate bonds did not cause any significant suppression for either method. The incorporation of 2'--methyl modifications suppressed fragmentation of the antisense strand of patisiran, which resulted in some gaps in sequence coverage. However, UVPD provided the highest sequence coverage when compared to a-EPD.
随着新型基于 RNA 的治疗方法的不断发展，对用于确认序列和修饰图谱的强大分析方法的需求也在加速增长。使用质谱法对修饰的核糖核酸进行特征描述具有挑战性，因为修饰核苷酸的诊断性片段可能会受到抑制，从而妨碍了完整的序列覆盖和修饰位置的确定。由于广泛的骨架断裂，紫外线光解（UVPD）在核酸特征描述方面显示出巨大的潜力。激活电子光脱附解离（a-EPD）也已被用作替代方法，以利用在 UVPD 中普遍存在的主导电荷还原途径，促进解离，并产生大量的片段离子。在这里，我们比较了高能碰撞激活（HCD）、使用 193nm 和 213nm 光子的 UVPD 以及 a-EPD 用于修饰核酸的自上而下测序，包括甲基化、硫代磷酸酯和锁核酸修饰的 DNA。这些修饰的存在改变了在 UVPD 和 a-EPD 中观察到的片段化途径，并且观察到广泛的骨架断裂，导致产生保留修饰的片段离子，从而可以对其进行精确定位。LNA 和 2'-甲氧基硫代磷酸酯修饰对 UVPD 的片段化有显著抑制作用，但对 a-EPD 没有抑制作用，而硫代磷酸酯键对这两种方法都没有显著抑制作用。2'-甲基修饰的掺入抑制了 patisiran 反义链的片段化，导致序列覆盖的一些缺口。然而，与 a-EPD 相比，UVPD 提供了最高的序列覆盖。
