RNase mapping by nucleobase-specific endonucleases combined with liquid chromatography/tandem mass spectrometry (LC/MS/MS) is a powerful analytical method for characterizing ribonucleic acids (RNAs). Endonuclease digestion of RNA yields products that contain a 3'-terminal phosphate group. MS/MS via collision-induced dissociation (CID) of these digestion products on a linear ion trap generates fragmentation pathways that include the loss of phosphoric acid (-H(3)PO(4); -98 u), which does not provide information about the sequence of the digestion products and can reduce ion abundance from other pathways that provide sequence information. Here we investigate the use of bacterial alkaline phosphatase (BAP) after RNase digestion to remove the 3'-terminal phosphate from all RNase digestion products prior to LC/MS/MS analysis. RNase digestion products lacking the 3'-phosphate were found to produce CID spectra with more consistent, high-abundance c- and y-type fragment ions as well as significantly more a-Base and w-type ions than digestion products retaining the 3'-phosphate. In this manner, RNase mapping with LC/MS/MS can provide more complete RNA sequence information from fragment ions of higher abundance that are easier to interpret and identify.
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