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Applications and Advantages of Stable Isotope Phosphate Labeling of RNA in Mass Spectrometry

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Abstract

Mass spectrometry (MS) has become an enabling technology for the characterization of post-transcriptionally modified nucleosides within ribonucleic acids (RNAs). These modified RNAs tend to be more challenging to completely characterize using conventional genomic-based sequencing technologies. As with many biological molecules, information relating to the presence or absence of a particular compound (i.e., qualitative measurement) is only one step in sample characterization. Additional useful information is found by performing quantitative measurements on the levels of the compound of interest in the sample. Phosphate labeling of modified RNAs has been developed by our laboratory to enhance conventional mass spectrometry techniques. By taking advantage of the mechanism of action of many ribonucleases (RNases), digesting RNA samples in the presence of 18O-labeled water generates an 18O-labeled 3′-phosphate in each digestion product. We describe the historical development of this approach, contrast this stable isotope labeling strategy with others used in RNA mass spectrometry, and provide examples of new analytical mass spectrometry methods that are enabled by phosphate labeling in this fashion.

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Fig. 1
Fig. 2

Reproduced with permission from Berhane et al. [28] Copyright 2003

Fig. 3

Reproduced with permission from Meng et al. [29] Copyright 2005

Fig. 4

Reproduced with permission from Li et al. [33] Copyright 2012

Fig. 5

Reproduced with permission from Li et al. [34] Copyright 2013

Fig. 6

Reproduced with permission from Wetzel et al. [35] Copyright 2014

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Acknowledgements

Financial support for our lab’s decade-long work in the area of stable isotope labeling of RNA has been generously supported by the National Science Foundation, including our current NSF support (CHE1507357). The generous support of the University of Cincinnati and the Rieveschl Endowment for these studies are also appreciated.

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Authors and Affiliations

  1. Rieveschl Laboratories for Mass Spectrometry, Department of Chemistry, University of Cincinnati, PO Box 210172, Cincinnati, OH, 45221-0172, USA

    Kayla Borland & Patrick A. Limbach

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  1. Kayla Borland
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  2. Patrick A. Limbach
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Correspondence to Patrick A. Limbach.

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This article is part of the Topical Collection “Phosphate Labeling and Sensing in Chemical Biology”; edited by Henning Jessen.

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Borland, K., Limbach, P.A. Applications and Advantages of Stable Isotope Phosphate Labeling of RNA in Mass Spectrometry. Top Curr Chem (Z) 375, 33 (2017). https://doi.org/10.1007/s41061-017-0121-z

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