rXXXX American Chemical Society A dx.doi.org/10.1021/ac200547y | Anal. Chem. XXXX, XXX, 000–000 ARTICLE pubs.acs.org/ac Characterization and Quantification of RNA Post-transcriptional Modifications Using Stable Isotope Labeling of RNA in Conjunction with Mass Spectrometry Analysis Sakharam P. Waghmare and Mark J. Dickman* Department of Chemical and Biological Engineering, ChELSI Institute, University of Sheffield, Mappin Street, Sheffield, S3 1JD, U.K. b S Supporting Information M ass spectrometry has emerged as an increasingly powerful tool for the identification and characterization of nucleic acids. Since this approach does not rely on amplification, post- transcriptional modifications are preserved and can be directly measured. Therefore, the approach can be used for the char- acterization of post-transcriptional RNA modifications 1,2 and potentially for the detection of DNA modifications. Prior to mass spectrometry analysis, the RNA of interest is routinely first purified using HPLC. 36 This often large biomo- lecule is subsequently digested with endonucleases into smaller oligoribonucleotides that are more amenable for chromato- graphic separation and intact mass measurements. 7,8 Additional sequence information from the oligoribonucleotides can be obtained using tandem mass spectrometry (MS/MS) via colli- sion-induced dissociation (CID) or postsource decay (PSD). 9 This approach has been successfully employed with a variety of configurations including liquid chromatography interfaced with electrospray ionization mass spectrometry (LC ESI MS) to characterize a wide range of RNAs. 2,8,1020 Matrix-assisted laser desorption ionization (MALDI) MS approaches have also been used to characterize RNA in conjunction with enzymatic diges- tions of the RNA. 21,22 However, the MS analysis of oligoribo- nucleotides generated via enzymatic digestions can produce a heterogeneous mixture, complicated by the presence of both the 2 0 3 0 -cyclic phosphate intermediate and 3 0 -phosphate digestion products. MS approaches have recently been developed for the identification and characterization of small RNAs from ribonu- cleoprotein complexes in conjunction with genome-orientated database searching. 23,24 Furthermore, electrophoretic separa- tions of RNA in conjunction with in-gel RNase digestion pro- cedures and LC MS analysis have also been successfully devel- oped for the analysis of RNA and the identification of post- transcriptional modifications. 25 Tandem mass spectrometry enables sequence characteriza- tion by selecting a precursor ion and rapid formation of fragment ions through collision with a neutral gas. Unfortunately, it is not straightforward to interpret the dissociation patterns of oligonu- cleotides. Compared to some other polymers, oligonucleotides typically generate complex spectra with a large number of frag- ment ions. Therefore, only limited success has been achieved in the de novo sequencing of oligonucleotides with this type of fragmentation. 2628 Recent developments in the optimization of tandem MS analysis of oligonucleotides have focused on the use of electron transfer dissociation (ETD) and infrared multiphoton dissociation (IRMPD) for the fragmentation of DNA and RNA oligonucleotides. 29,30 A wide range of mass spectrometers has been employed for the characterization of RNA using the above approaches. 7 However, Received: March 10, 2011 Accepted: May 3, 2011 ABSTRACT: Mass spectrometry has emerged as an increas- ingly powerful tool for the identification and characterization of nucleic acids, in particular RNA post-transcriptional modifica- tions. High mass accuracy instrumentation is often required to discriminate between compositional isomers of oligonucleo- tides. We have used stable isotope labeling ( 15 N) of E. coli RNA in conjunction with mass spectrometry analysis of the combined heavy- and light-labeled RNA for the identification and quanti- fication of oligoribonucleotides and post-transcriptional mod- ifications. The number of nitrogen atoms in the oligoribonucleotide and fragment ions can readily be determined using this approach, enabling the discrimination between potential compositional isomers without the requirement of high mass accuracy mass spectrometers. In addition, the identification of specific fragment ions in both the unlabeled and labeled oligoribonucleotides can be used to gain further confidence in the assignment of RNA post-transcriptional modifications. Using this approach we have identified a range of post-transcriptional modifications of E. coli 16S rRNA. Furthermore, this method facilitates the rapid and accurate quantification of oligoribonucleotides, including cyclic phosphate intermediates and missed cleavages often generated from RNase digestions.