Intact microRNA analysis using high resolution mass spectrometry.
Intact microRNA analysis using high resolution mass spectrometry.
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DOI：
10.1007/s13361-013-0759-x
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发表时间：
2014-01
影响因子：
3.2
通讯作者：
Pitteri, Sharon J.
中科院分区：
文献类型：
--
作者：
Kullolli, Majlinda;Knouf, Emily;Arampatzidou, Maria;Tewari, Muneesh;Pitteri, Sharon J.
MicroRNAs(MiRNAs)是一种小的单链非编码RNA，它在转录后调节基因表达，在各种细胞过程和疾病的调节中发挥关键作用。分析miRNAs的新工具将增加对这类分子的生理起源和生物学功能的理解。在这项研究中，我们通过概念验证实验来研究高分辨率质谱学在miRNAs分析中的应用。我们证明了质谱学能够分辨和分离混合物中的miRNAs和相应的3‘变异体。来自各种miRNAs的单同位素去质子峰的质量精度在低ppm范围内。我们比较了碰撞诱导解离(CID)和高能碰撞解离(HCD)对miRNA的碎片，后者产生了两种方法相似的序列覆盖率，但HCD和CID产生了额外的碎片。我们测量了负载到C18柱上的miRNA的线性动态范围、检测限和定量限。最后，我们探索了在线LC-MS期间对miRNA的MS/MS光谱的数据依赖获取的使用，并证明了多个电荷状态可以被碎裂，在色谱时间尺度上产生几乎全序列的miRNA覆盖。我们的结论是，高分辨率质谱仪可以分离和测量混合物中的miRNAs，标准的LC-MS装置可以用于这些分子的在线分析。
MicroRNAs (miRNAs) are small single-stranded non-coding RNAs that post-transcriptionally regulate gene expression, and play key roles in the regulation of a variety of cellular processes and in disease. New tools to analyze miRNAs will add understanding of the physiological origins and biological functions of this class of molecules. In this study we investigate the utility of high resolution mass spectrometry for the analysis of miRNAs through proof-of-concept experiments. We demonstrate the ability of mass spectrometry to resolve and separate miRNAs and corresponding 3′ variants in mixtures. The mass accuracy of the monoisotopic deprotonated peaks from various miRNAs is in the low ppm range. We compare fragmentation of miRNA by collision-induced dissociation (CID) and by higher-energy collisional dissociation (HCD) which yields similar sequence coverage from both methods but additional fragmentation by HCD versus CID. We measure the linear dynamic range, limit of detection, and limit of quantitation of miRNA loaded onto a C18 column. Lastly we explore the use of data dependent acquisition of MS/MS spectra of miRNA during online LC-MS and demonstrate that multiple charge states can be fragmented, yielding nearly full sequence coverage of miRNA on a chromatographic time scale. We conclude that high resolution mass spectrometry allows the separation and measurement of miRNAs in mixtures and a standard LC-MS setup can be adapted for online analysis of these molecules.
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microRNA分析：方法和考虑因素。
DOI：
10.1038/nrg3198
发表时间：
2012-04-18
期刊：
Nature reviews. Genetics
影响因子：
--
作者：
Pritchard CC;Cheng HH;Tewari M
通讯作者：
Tewari M
影响因子：
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作者：
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DOI：
10.1016/j.jasms.2009.10.010
发表时间：
2010-02-01
影响因子：
3.2
作者：
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通讯作者：
Breuker, Kathrin
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影响因子：
44.1
作者：
Chen, Xi;Ba, Yi;Zhang, Chen-Yu
通讯作者：
Zhang, Chen-Yu
影响因子：
2
作者：
GREIG, M;GRIFFEY, RH
通讯作者：
GRIFFEY, RH