Intact MicroRNA Analysis Using High Resolution Mass Spectrometry
- 1.2k Downloads
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.
Key wordsmicroRNA Oligonucleotide RNA
The authors are grateful for support from the 2012 American Society for Mass Spectrometry (ASMS) Research Award (to S.J.P.), the Canary Foundation (to S.J.P. and M.T.), and US National Institutes of Health Transformative R01 grant R01DK085714 (to M.T.). The authors acknowledge Dr. Jason Hogan and Mr. Jake Kennedy for thoughtful discussions. and Emily Gallichotte for technical assistance.
- 1.Chen, X., Ba, Y., Ma, L., Cai, X., Yin, Y., Wang, K., Guo, J., Zhang, Y., Chen, J., Guo, X., Li, Q., Li, X., Wang, W., Wang, J., Jiang, X., Xiang, Y., Xu, C., Zheng, P., Zhang, J., Li, R., Zhang, H., Shang, X., Gong, T., Ning, G., Zen, K., Zhang, C.Y.: Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases. Cell Res. 18, 997–1006 (2008)CrossRefGoogle Scholar
- 18.Olsen, J.V., Schwartz, J.C., Griep-Raming, J., Nielsen, M.L., Damoc, E., Denisov, E., Lange, O., Remes, P., Taylor, D., Splendore, M., Wouters, E.R., Senko, M., Makarov, A., Mann, M., Horning, S.: A dual pressure linear ion trap Orbitrap instrument with very high sequencing speed. Mol. Cell Proteom. 8, 2759–2769 (2009)CrossRefGoogle Scholar