SEQUENCE INFORMATION FROM 42-108-MER DNAS (COMPLETE FOR A 50-MER) BY TANDEM MASS-SPECTROMETRY

Complete sequence information for an ''unknown'' 50-mer DNA and extens ive sequence verification for another 50-mer and 42-, 51-, 55-, 60-, 7 2-, 100-, and 108-mer DNAs is obtained by electrospray ionization/Four ier transform mass spectrometry that supplies 10-100x higher accuracy and resolving-power data using nozzle-skimmer (NS), collisionally acti vated, and infrared multiphoton dissociation (IRMPD). In addition to t he previously recognized 3'- and 5'-terminal (w and a) ions, internal ions (i) and MS/MS/MS of fragment ions provide unique structural infor mation across the DNA. NS dissociation can also yield other new backbo ne cleavages (forming b, c, d, and r ions) that provide extensive 5'-e nd information. These spectra indicate that loss of the base T rarely triggers formation of w, a, or i fragment ions, a correlation of furth er sequencing utility. Point mutation screening is demonstrated using a modified 50-mer unknown; a 9.04 (theory 9.01) decrease in the molecu lar weight (M(r)) value indicates A --> while three IRMPD fragment ion s pinpoint this mutation at base 27. Introduction (measurement time <1 min) of 8 x 10(-16) mol of the 50-mer gave an M(r) value with only a 0.2 error.