De novo sequencing of short interfering ribonucleic acids facilitated by use of tandem mass spectrometry with ion mobility spectrometry

Abstract

The use of RNAi for new therapeutics is becoming more widespread. To improve the development and quality control of such materials there is a need for rapid, accurate and meaningful analyses. Here, the use of negative ion nano-electrospray ionisation tandem mass spectrometry with ion mobility spectrometry (nESI-MS/MS-IMS-MS) is shown to simplify data interpretation and lead to higher sequence coverage. A set of 20-nucleotide RNA molecules was analysed using nESI-MS/MS and their sequences determined manually with the aid of the Simple Oligonucleotide Sequencer (SOS) program. The RNAs were also analysed using nESI-MS/MS-IMS-MS. This incorporates an extra step involving travelling-wave IMS separation of the product ions into groups according to the number of charges that the ions carry. Following this, the RNA sequences were determined from the separated groups of ions. nESI-MS/MS collision-induced dissociation of the RNA sequences produced w, y, a-(Base) and c product ions. Sequence determination resulted in incomplete coverage with bases in the centre of the sequences being unidentifiable because of the plethora of overlapping ions. Sequencing carried out from the nESI-MS/MS-IMS-MS data, whereby individual product ion spectra arising only from ions carrying the same charge were generated, gave full sequence coverage for each nucleotide (except y1 ) with assignment confirmation from a minimum of four different product ions. Using nESI-MS/MS-IMS-MS to analyse a number of 20-nucleotide RNA molecules produced full sequence coverage with 100% accuracy, in addition to molecular mass confirmation. This method has the potential for automation for higher sample throughput and thus constitutes a robust approach for the quality control of RNAs in therapeutics.