Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection - PubMed (original) (raw)

Improving CMC-derivatization of pseudouridine in RNA for mass spectrometric detection

Anita Durairaj et al. Anal Chim Acta. 2008.

Abstract

A protocol that utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and N-cyclohexyl-N'-beta-(4-methylmorpholinium)ethylcarbodiimide (CMC) derivatization to detect the post-transcriptionally modified nucleoside, pseudouridine, in RNA has been optimized for RNase digests. Because pseudouridine is mass-silent (i.e., the mass of pseudouridine is the same as the mass of uridine), after CMC-derivatization and alkaline treatment, all pseudouridine residues exhibit a mass shift of 252 Da that allows its presence to be easily detected by mass spectrometry. This protocol is illustrated by the direct MALDI-MS identification of pseudouridines within Escherichia coli tRNA(TyrII) starting from microgram amounts of sample. During this optimization study, it was discovered that the post-transcriptionally modified nucleoside 2-methylthio-N(6)-isopentenyladenosine, which is present in bacterial tRNAs, also retains a CMC unit after derivatization and incubation with base. Thus, care must be exercised when applying this MALDI-based CMC-derivatization approach for pseudouridine detection to samples containing transfer RNAs to minimize the misidentification of pseudouridine.

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Figures

Figure 1

Figure 1

(a) MALDI mass spectrum of r(AUGCAUGC) after derivatization with CMC p-tosylate in 50 mM Tris, 4 mM EDTA, 7M urea (pH 8.3) overnight at 37 °C. The number of CMC adducts are noted on each mass spectrum. (b) MALDI mass spectrum of r(AUGCAUGC) after incubating the sample in Figure 1a in 50 mM NH4OH (pH 10.4) for 60 min at 80 °C. These incubation conditions allow for the complete release of CMC without degrading the oligonucleotide.

Figure 2

Figure 2

(a) MALDI mass spectrum of E. coli tRNATyrII digested with RNase T1. (b) MALDI mass spectrum of the CMC-derivatized RNase T1 digest of E. coli tRNATyrII after alkaline treatment. Table 1 lists the RNase T1 digestion products including those which retain CMC after alkaline treatment along with tRNA signature digestion products identified from contaminating E. coli tRNAs in the sample.

Figure 3

Figure 3

(a) MALDI mass spectrum of E. coli tRNATyrII digested with RNase A. (b) MALDI mass spectrum of the CMC-derivatized RNase A digest of E. coli tRNATyrII after alkaline treatment. Table 2 lists the RNase A digestion products including those which retain CMC after alkaline treatment along with tRNA signature digestion products identified from contaminating E. coli tRNAs in the sample.

Figure 4

Figure 4

MALDI mass spectrum of the products arising from a 2 h snake venom phosphodiesterase digestion of the RNase A digestion product, A[ms2i6A]AACp, from E. coli tRNATyrII after CMC derivatization and alkaline treatment. Underivatized A[ms2i6A]AACp is also present in this sample.

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