Minimizing Base Loss and Internal Fragmentation in Collisionally Activated Dissociation of Multiply Deprotonated RNA
Minimizing Base Loss and Internal Fragmentation in Collisionally Activated Dissociation of Multiply Deprotonated RNA
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DOI：
10.1016/j.jasms.2009.10.010
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发表时间：
2010-02-01
影响因子：
3.2
通讯作者：
Breuker, Kathrin
中科院分区：
文献类型：
--
作者：
Taucher, Monika;Rieder, Ulrike;Breuker, Kathrin
近年来，发现了一类具有重要细胞功能的非蛋白质编码核糖核酸（ncRNA）。对于生物分子研究和药物开发特别感兴趣的是参与基因调控的小ncRNA，例如小干扰RNA（21-28 nt）、前微RNA（70-80 nt）或核糖开关（34-200 nt）。迄今为止，通过自上而下质谱法对RNA进行的从头测序仅限于由多达20个核苷酸组成的RNA。我们在这里报告完整的序列覆盖34 nt RNA（10.9 kDa），沿着与30的32个可能的互补离子对碰撞激活解离（CAD）实验。最小化不期望的碱基损失和内部碎裂的关键是最小化来自初级主链裂解的碎片离子的内能。这可以通过初级碎片离子的碰撞冷却和选择相对低的负净电荷（约-0.2/nt）的前体离子来实现。（J Am Soc Mass Spectrom 2010，21，278-285）（C）2010年美国质谱学会
In recent years, new classes of nonprotein-coding ribonucleic acids (ncRNAs) with important cellular functions have been discovered. Of particular interest for biomolecular research and pharmaceutical developments are small ncRNAs that are involved in gene regulation, such as small interfering RNAs (21-28 nt), pre-microRNAs (70-80 nt), or riboswitches (34-200 nt). De novo sequencing of RNA by top-down mass spectrometry has so far been limited to RNA consisting of up to similar to 20 nt. We report here complete sequence coverage for 34 nt RNA (10.9 kDa), along with 30 out of 32 possible complementary ion pairs from collisionally activated dissociation (CAD) experiments. The key to minimizing undesired base loss and internal fragmentation is to minimize the internal energy of fragment ions from primary backbone cleavage. This can be achieved by collisional cooling of primary fragment ions and selection of precursor ions of relatively low negative net charge (about -0.2/nt). (J Am Soc Mass Spectrom 2010, 21, 278-285) (C) 2010 American Society for Mass Spectrometry