Improved oligonucleotide sequencing by alkaline phosphatase and exonuclease digestions with mass spectrometry

The combination of exonuclease digestion and mass spectrometry has been wid ely used for sequencing oligonucleotides. During an exonuclease digestion, rapid buildup in the concentration of nucleotides produces strong signal of nucleotide cluster ions in electrospray ionization-mass spectrometry, espe cially for oligonucleotides with greater than 25 bases. This leads to poor signal/noise ratio in the reconstructed molecular weight spectra of late di gestion products due to artifact peaks from nucleotide cluster ions. Here w e report a procedure that eliminates the effect of the cluster ions. In thi s method, alkaline phosphatase is added with snake venom phosphodiesterase to the oligonucleotide solution to convert the interfering nucleotides into noninterfering nucleosides, and the collision-induced dissociation spectru m of the dimeric oligonucleotide at the end of the digestion is obtained to determine the sequence of the last two bases at the 6 ' -terminus of the o ligonucleotide. With this approach, the signal/noise ratio of the reconstru cted molecular weight spectrum is greatly improved for relatively large oli gonucleotides, and only a single digestion is needed for sequencing. (C) 20 01 Academic Press.