Addressing the Determinants of Ambiguous Assignments Incurred by the Mass Spectrometry Sequence Analysis of Large...

Common mass spectrometric approaches for the characterization of large RNAs involve cleaving the initial strand into smaller sections amenable to mass mapping and gas-phase sequencing. These approaches are plagued by the significant loss of information associated with the production of ambiguous isomeric/isobaric products by the classic nucleotide-specific endonucleases. In this report, we investigated the intrinsic determinants of such ambiguities and evaluated the ability of alternative experimental strategies to minimize their impact. Simulating the RNase T1 digestion of test analytes spanning up to 1869 nt showed that the number and spacing of susceptible sites determined the possible isomeric/isobaric status, but product size was the most important factor. The outcome of limited digestion and time-course analyses showed that promoting missed cleavages could effectively increase the average size of digestion products without exceeding the range accessible by gas-phase sequencing, which translated into significant reductions of ambiguous assignments. Indeed, these approaches yielded mass mapping and sequencing coverages in the 92-98% and 76-92% ranges for the test analytes. The study demonstrated that while the intrinsic determinants of mass degeneracy cannot be eliminated, their impact can be effectively managed through careful experimental design.