Sequencing 50-mer DNAs Using Electrospray Tandem Mass Spectrometry and Complementary Fragmentation Methods

摘要

Sequencing DNA with < 100 bases by the common enzymatic dideoxy cDNA technique is more complicated than it is for larger DNA, so that chemical degradation is usually employed. However, the latter requires ～50 pmol of radioactive ~(32)P end-labeled material, six chemical steps, electrophoretic separation, and film exposure. For small nucleotides (≤ 14-mer) the combination of electrospray ionization (ESI) and Fourier transform (FT) mass spectrometry (MS) is far faster and more sensitive; dissociation products of multiply-charged ions measured at high (10~5) resolving power (RP)~8 represent consecutive backbone cleavages providing the full sequence in <1 min on subpicomole samples. For molecular weight (MW) measurements, ESI/MS has recently been extended to larger strands, including a 132-mer and a single ion of 10~8 Da T4 phage DNA; ESI/FTMS of 50-, 72-, and 100-mers gave molecular mass values of 1 Da accuracy. Although for smaller DNA (≤ 25-mers) nozzle-skimmer dissociation (NS) and infrared multiphoton dissociation (IRMPD) of ESI ions gave essentially the same sequence information, here we demonstrate for a 50-mer that NS and IRMPD induce different yet complementary fragmentation patterns providing nearly complete sequence information.