SEQUENCING REGULAR AND LABELED OLIGONUCLEOTIDES USING ENZYMATIC DIGESTION AND IONSPRAY MASS-SPECTROMETRY

A method using a combination of enzymatic digestion and ionspray mass spectrometry (MS) was developed for sequencing oligodeoxynucleotides ( ODNs) containing more than 20 bases. Phosphodiesterase (PDE) digestion of ODNs produced truncated ODNs whose molecular weights (MWs) were de termined by ionspray MS, It was demonstrated that reconstruction of MW spectra over a large MW range produced easy-to-read sequence ladders similar to those obtained using matrix-assisted laser desorption/ioniz ation time-of-flight mass spectrometry (MALDI-TOF-MS). Sample and enzy me cleanup, digestion control, and 1MW reconstruction were found to be crucial factors. For regular ODNs, both 5'- and 3'-PDE digestions are needed for complete sequencing. Late in the time course of PDE digest ions, 5'-nucleoside monophosphates were found to produce artifactual p eaks in the reconstructed MW spectra, and a table correlating base com positions and MS ions was compiled to handle such situations. For labe led ODNs, it is necessary to use collision-induced dissociation-tandem mass spectrometry (CID-MS/MS) for complete sequence determination. Se quencing of regular 22-mer and labeled 18-mer ODNs was demonstrated in this work. (C) 1998 Academic Press.