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

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.