背景:该研究解决了当商业标准不可用于生成参考光谱文库时鉴定RNA转录后修饰的挑战。它建议使用同源核碱基和脱氧核糖核苷作为替代参考光谱文库,以帮助鉴定修饰的核糖核苷,并在标准品不可用时将其与位置异构体区分开。
方法:核糖核苷的全套,使用与OrbitrapEclipseTribrid质谱仪耦合的高性能纳米流动液相色谱法分析脱氧核糖核苷和核碱基标准品。使用靶向MS2和中性丢失触发的MS3方法,从同源核碱基和脱氧核糖核苷构建光谱文库,和碰撞能量进行了优化。使用这些文库鉴定修饰的核糖核苷及其位置异构体的可行性通过光谱片段化模式的比较来评估。
结果:我们的分析表明,MS2和中性丢失触发的MS3方法都产生了丰富的光谱,在核糖核苷之间具有相似的片段化模式,脱氧核糖核苷和核碱基。此外,我们证明了核碱基和脱氧核糖核苷的光谱,在优化的碰撞能量下产生,与修饰的核糖核苷表现出足够的相似性,以使其能够用作参考光谱,以准确鉴定核糖核苷家族中的位置异构体。
结论:该研究证明了利用同源核碱基和脱氧核糖核苷作为可互换的参考光谱文库鉴定修饰的核糖核苷及其位置异构体的有效性。这种方法为克服商业标准不可用带来的限制提供了有价值的解决方案。通过质谱增强RNA转录后修饰的分析。
BACKGROUND: The study addresses the challenge of identifying RNA post-transcriptional modifications when commercial standards are not available to generate reference spectral libraries. It proposes employing homologous nucleobases and deoxyribonucleosides as alternative reference spectral libraries to aid in identifying modified
ribonucleosides and distinguishing them from their positional isomers when the standards are unavailable.
METHODS: Complete sets of ribonucleoside, deoxyribonucleoside and nucleobase standards were analyzed using high-performance nano-flow liquid chromatography coupled to an Orbitrap Eclipse Tribrid mass spectrometer. Spectral libraries were constructed from homologous nucleobases and deoxyribonucleosides using targeted MS2 and neutral-loss-triggered MS3 methods, and collision energies were optimized. The feasibility of using these libraries for identifying modified
ribonucleosides and their positional isomers was assessed through comparison of spectral fragmentation patterns.
RESULTS: Our analysis reveals that both MS2 and neutral-loss-triggered MS3 methods yielded rich spectra with similar fragmentation patterns across
ribonucleosides, deoxyribonucleosides and nucleobases. Moreover, we demonstrate that spectra from nucleobases and deoxyribonucleosides, generated at optimized collision energies, exhibited sufficient similarity to those of modified
ribonucleosides to enable their use as reference spectra for accurate identification of positional isomers within ribonucleoside families.
CONCLUSIONS: The study demonstrates the efficacy of utilizing homologous nucleobases and deoxyribonucleosides as interchangeable reference spectral libraries for identifying modified
ribonucleosides and their positional isomers. This approach offers a valuable solution for overcoming limitations posed by the unavailability of commercial standards, enhancing the analysis of RNA post-transcriptional modifications via mass spectrometry.