Minimizing base loss and internal fragmentation in collisionally activated dissociation of multiply deprotonated RNA

In recent years, new classes of nonprotein-coding ribonucleic acids (ncRNAs) with important cellular functions have been discovered. Of particular interest for biomolecular research and pharmaceutical developments are small ncRNAs that are involved in gene regulation, such as small interfering RNAs (21-28 nt), pre-microRNAs (70-80 nt), or riboswitches (34-200 nt). De novo sequencing of RNA by top-down mass spectrometry has so far been limited to RNA consisting of up to approximately 20 nt. We report here complete sequence coverage for 34 nt RNA (10.9 kDa), along with 30 out of 32 possible complementary ion pairs from collisionally activated dissociation (CAD) experiments. The key to minimizing undesired base loss and internal fragmentation is to minimize the internal energy of fragment ions from primary backbone cleavage. This can be achieved by collisional cooling of primary fragment ions and selection of precursor ions of relatively low negative net charge (about -0.2/nt).