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 extensive sequence verification for another 50-mer and 42-, 51-, 55-, 60-, 72-, 100-, and 108-mer DNAs is obtained by electrospray ionization/Fourier transform mass spectrometry that supplies 10−100× higher accuracy and resolving-power data using nozzle-skimmer (NS), collisionally activated, and infrared multiphoton dissociation (IRMPD). In addition to the previously recognized 3‘- and 5‘-terminal (w and a) ions, internal ions (i) and MS/MS/MS of fragment ions provide unique structural information across the DNA. NS dissociation can also yield other new backbone cleavages (forming b, c, d, and r ions) that provide extensive 5‘-end information. These spectra indicate that loss of the base T rarely triggers formation of w, a, or i fragment ions, a correlation of further sequencing utility. Point mutation screening is demonstrated using a modified 50-mer unknown; a 9.04 (theory 9.01) decrease in the molecular weight ( M r ) value indicates A → T, while three IRMPD fragment ions pinpoint this mutation at base 27. Introduction (measurement time <1 min) of 8 × 10 - 16 mol of the 50-mer gave an M r value with only a 0.2 error.