Peter Williams, Chau-Wen Chou, David Dogruel, Jennifer Krone, Kathy Lewis, Randall Nelson
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287-1604
There are three potential roles for mass spectrometry relevant to the Human Genome Project:
a) The most obvious role is that on which all groups have been focussing - development of an alternative, faster sequence ladder readout method to speed up large-scale sequencing. Progress here has been difficult and slow because the mass spectrometry requirements exceed the current capabilities of mass spectrometry even for proteins, and DNA presents significantly more difficulty than proteins. We have shown previously that pulsed laser ablation of DNA from frozen aqueous films has the potential to yield sequence-quality mass spectra, but that ionization in this approach is erratic and uncontrollable. We are focussing on developing ionization methods using ion (or electron) attachment to vapor-phase DNA (ablated from ice films) in an electric field-free environment; results of this approach will be reported.
b) Mass spectrometry may not ultimately compete favorably in speed with large-scale multiplexing of conventional or near-term technologies such as capillary electrophoresis. However, as the Genome project nears completion there will be an increasing need for rapid small-scale DNA analysis, where the multiplex advantage will not be so great and mass spectrometry could play a more significant role there. With this in mind we are looking at ways to speed up the overall mass spectrometric analysis, e.g. simple rapid cleanup of sequence mixtures, and at generation of short sequence ladders by exopeptidase digestion.
c) Given the genome data base(s) at the completion of the project, with rapid search capability, a need will arise for comparably rapid generation of search input data to identify often very small quantities of proteins isolated from biochemical investigations. With this in mind we have developed extremely rapid enzyme digestion techniques optimized for mass spectrometric readout, using endopeptidases covalently coupled directly to the mass spectrometer probe tip. The elimination of autolysis and transfer losses allows rapid (few minute) endopeptidase digestion and mass analysis of as little as 1 picomole of protein, leading to an umbiguous database identification. An alternative search procedure uses partial amino-acid sequence information. With the added use of exopeptidases to generate a peptide ladder sequence in the mass spectrum of the endopeptidase digest, on the order of a dozen residues of internal sequence can be generated in a total analysis time of 20 minutes or less, again using only picomoles of sample.