High-resolution ion mobility spectrometry-mass spectrometry of isomeric/isobaric ribonucleotide variants.
High-resolution ion mobility spectrometry-mass spectrometry of isomeric/isobaric ribonucleotide variants.
复制标题
DOI：
10.1002/jms.4465
复制
发表时间：
2020-03
期刊：
影响因子：
--
通讯作者：
Fabris D
中科院分区：
文献类型：
--
作者：
Kenderdine T;Nemati R;Baker A;Palmer M;Ujma J;FitzGibbon M;Deng L;Royzen M;Langridge J;Fabris D
在本报告中，我们探讨了循环离子淌度 (cIM) 质谱分析在 RNA 异构体转录后修饰分析中的优势。标准甲基胞苷样品最初用于测试正确区分具有相同元素组成和分子质量的不同结构的能力。单独分析时，分析物显示出由修饰甲基在共同胞苷支架上的不同位置决定的特征到达时间 (tD)。在混合分析中，各个信号的宽度导致了显着的重叠，这最初阻碍了它们在 tD 尺度上的分辨率。四种异构体的分离是通过增加通过 cIM 装置的次数来实现的，这使得能够完全区分与非常微妙的结构变化相关的特征离子淌度行为。将 cIM 设备放置在质量选择四极杆和飞行时间分析仪之间，使我们能够对每个离子群进行气相激活，这些离子群首先根据共同的质荷比进行分离，然后根据不同的离子淌度行为进行分离。观察到的碎片模式证实了各种异构体的结构，从而证实了用特定碎片数据补充独特的 tD 信息以实现更严格的分析物鉴定的好处。通过分析总 RNA 提取物的核酸外切酶消化获得的天然单核苷酸混合物，进一步测试了这些能力。特别是，cIM 分离和迁移后解离的结合使我们能够确定 HeLa 细胞整个转录组中存在的甲基胞苷和甲基腺嘌呤成分的组成。因此，我们预计该技术不仅有利于需要确定 RNA 修饰的身份和表达水平的表观转录组学研究，而且有利于涉及分析可能含有异构体/同量异位物种子集的天然提取物的代谢组学研究。
In this report, we explored the benefits of cyclic ion mobility (cIM) mass spectrometry in the analysis of isomeric post-transcriptional modifications of RNA. Standard methyl-cytidine samples were initially utilized to test the ability to correctly distinguish different structures sharing the same elemental composition, and thus molecular mass. Analyzed individually, the analytes displayed characteristic arrival times (tD) determined by the different positions of the modifying methyl groups onto the common cytidine scaffold. Analyzed in mixture, the widths of the respective signals resulted in significant overlap that initially prevented their resolution on the tD scale. The separation of the four isomers was achieved by increasing the number of passes through the cIM device, which enabled to fully differentiate the characteristic ion mobility behaviors associated with very subtle structural variations. The placement of the cIM device between the mass-selective quadrupole and the time-of-flight analyzer allowed us to perform gas-phase activation of each of these ion populations, which had been first isolated according to a common mass-to-charge ratio, and then separated on the basis of different ion mobility behaviors. The observed fragmentation patterns confirmed the structures of the various isomers, thus substantiating the benefits of complementing unique tD information with specific fragmentation data to reach more stringent analyte identification. These capabilities were further tested by analyzing natural mono-nucleotide mixtures obtained by exonuclease digestion of total RNA extracts. In particular, the combination of cIM separation and post-mobility dissociation allowed us to establish the composition of methyl-cytidine and methyl-adenine components present in the entire transcriptome of HeLa cells. For this reason, we expect that this technique will benefit not only epitranscriptomics studies requiring the determination of identity and expression levels of RNA modifications, but also metabolomics investigations involving the analysis of natural extracts that may possibly contain subsets of isomeric/isobaric species.
登录
查看更多内容
影响因子：
7.3
作者：
Limbach, Patrick A.;Paulines, Mellie June
通讯作者：
Paulines, Mellie June
影响因子：
14.9
作者：
Dunin-Horkawicz, Stanislaw;Czerwoniec, Anna;Gajda, Michal J.;Feder, Marcin;Grosjean, Henri;Bujnicki, Janusz M.
通讯作者：
Bujnicki, Janusz M.
影响因子：
14.9
作者：
KOWALAK, JA;POMERANTZ, SC;MCCLOSKEY, JA
通讯作者：
MCCLOSKEY, JA
影响因子：
4.5
作者：
Kirpekar, F;Douthwaite, S;Roepstorff, P
通讯作者：
Roepstorff, P
影响因子：
7.4
作者：
Kune, Christopher;Far, Johann;De Pauw, Edwin
通讯作者：
De Pauw, Edwin