DNA SEQUENCE LADDER READOUT BY MASS SPECTROMETRY

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A new mass spectrometric approach is proposed for rapid readout of Sanger dideoxy DNA sequence ladders. Ions will be generated using the relatively new technique known as Massive Cluster Impact (MCI) desorption. MCI generates vapor-phase ions from solid or liquid (solution) targets by the hypervelocity impact of massive, highly-charged glycerol or water dusters produced in vacuum by an electrospray process. The hypervelocity cluster impact has been shown to shock-heat and desorb massive biomolecular ions with a low degree of internal excitation and consequently long lifetimes, allowing mass spectrometric detection with good mass resolution. Ions arc generated in vacuum, allowing efficient transport into the mass spectrometer and minimizing sample size requirements. The initial extent of multiple charging in the desorbed ions is relatively low. To accomplish these studies, a cluster ion source and sample introduction system will be interfaced to a time-of-flight mass spectrometer with pulsed orthogonal ion extraction and an ion reflector for high mass resolution. Methods to control the degree of charging will be investigated: these include varying the cluster charge and energy, and doping the target and/or the cluster feed liquid with proton donors or acceptors to perturb the competition of the DNA analyte for charged solution species or the excess protons carried into the surface by the impacting cluster. Facile charge control which minimizes multiple charging should allow optimized analyses of multicomponent DNA sequence ladder mixtures. To investigate the competing roles of cluster charge and ionization equilibria in determining analyte ion charge multiplicity, experiments will also be performed in which analyte-containing clusters are electrosprayed and desolvated by impact on clean solid and liquid targets. MCI desorption of DNA from affinity capture surfaces will be investigated, in order to facilitate sample cleanup and minimize sample size requirements.