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

Abstract

A protocol that utilizes matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and N-cyclohexyl-N′-β-(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 MALDIbased CMC-derivatization approach for pseudouridine detection to samples containing transfer RNAs to minimize the misidentification of pseudouridine.

Pseudouridine in the Anticodon of Escherichia coli tRNATyr(QΨA) Is Catalyzed by the Dual Specificity Enzyme RluF

“…However, the mass of detected product ions shifted to two carbodiimide units from c 9 (m/z 1687.7 3 m/z 1813.3) onward and y 8 (m/z 1429.0 3 m/z 1554.6) onward (supplemental Table S1. These data are in contrast with a previous finding (28) where RNase A and snake venom phosphodiesterase-digested tRNA Tyr was analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) after CMCT derivatization. Those earlier data also showed that two carbodiimide units were added to the anticodon region of tRNA Tyr .…”

“…Prior reports discussed the possibility of an additional pseudouridine (31) or off-target derivatization (28), but until now, those possibilities remained uninvestigated. To clarify this open question, we conducted an exhaustive examination of E. coli tRNA Tyr using multiple pseudouridine detection strategies.…”

“…Chemical Derivatization-For CMCT derivatization, the RNA digest was derivatized with 10 l of CMCT (10 mg/ml) solution made with 50 mM Tris-HCl, pH 8.3, 4 mM EDTA, and 7 M urea (28). This concentration was found to be optimal based on the independent LC-MS/MS analysis of ⌿-containing (⌿GG, A⌿CAG, and UAAC⌿A⌿GACG) and ⌿-lacking (UGG, AUCAG, and UAACUAUGACG) oligonucleotides (Dharmacon, GE Healthcare) following derivatization and alkali treatment (10 l of 50 mM ammonium bicarbonate, pH 10.4, at 75°C for 90 min).…”

“…Prior reports discuss the possibility of an additional pseudouridine (31) or off-target derivatization (28), but until now, those possibilities remained uninvestigated. Here we have identified the additional pseudouridine derivatization site as residing at position 35 in the anticodon.…”

“…Recently, CMCT-based derivatization has been adapted to high throughput next generation sequencing to detect dynamic pseudouridylations in mRNAs (24,25). Mass spectrometrybased RNA modification mapping in combination with chem-ical derivatization is another site-specific strategy (26) wherein a base-specific ribonuclease generates a mixture of oligoribonucleotides whose pseudouridines are chemically derivatized with either CMCT (27)(28)(29) or acrylonitrile (30,31) for detection.…”