1. Reflectron TOF-MS is often used to improve mass resolution since the velocity distribution that spoils mass resolution in linear time-of-flight mass spectrometers can be compensated. However, for large DNA fragment measurements, the use of reflectron time-of-flight does not significantly improve mass resolution. It does, however, reduce the signal level drastically due to the limited lifetimes of DNA ions (18) Thus, a linear time-of-flight mass spectrometer is still preferred.

2. In general, electrospray mass spectrometry can exhibit better mass resolution than TOF-MS. However, an electrospray mass spectrometer is not suitable for measuring several bio-molecules simultaneously, since each molecule tends to give several peaks due to the multiply charged ions. On the other hand, when only one short tandem repeat allele is to be analyzed, an electrospray mass spectrometer can give better mass resolution for determining the sizes of DNAs. However, sample impurity (i.e., salt contamination) is very critical for using electrospray mass spectrometry for DNA measurements. If electrospray mass spec-trometry is to be used, more extensive DNA purification will be necessary.

3. Instead of a mixture of 3-hydroxypicolinic acid, picolinic acid, and ammonium fluoride, it is also possible to use a mixture of 0.2 M 2,3,4-trihydroxyacetophenone, 0.2 M 2,4,5-trihydroxyacetophenone, and 0.3 M ammonium citrate dibasic of equal volume (19) as the matrix. In general, the mass resolution is better with this alternate matrix but the ion signal amplitude is less for large DNA fragments, compared to the 3-HPA solution.

4. Three characteristics of the matrix are important in successful MALDI-MS experiments: 1) The matrix must incorporate the analyte in a way that does not break bonds or otherwise compromise the integrity of the intra-DNA binding; 2) the matrix must have strong absorption at the chosen laser wavelength; and 3) the matrix must provide a ready source of charge, which can be readily transferred to the DNA molecules. This is critically important, as the interaction between analyte molecules and crystals might be the key that determines the usefulness of a matrix material for MALDI.

5. Our results of MALDI for both DRPLA and HD samples show that mass spectrometry can be used for the accurate determination of the number of CAG repeats. Since the time needed for mass spectrometric analysis can be much shorter than the gel electrophoretic method, MALDI has the potential to become a valuable tool for the quick diagnosis of these dominantly inherited neurodegenerative disorders as well as a useful methodology for the detection of repeat-containing genes in general. We expect the same technology can be applied to other repeat expansion diseases. Microsatellites, which often contain tandem repeats, are useful markers for many genetic loci. Thus, we expect that MALDI-TOF-MS will be useful for the analysis of microsatellite markers as well. However, expansion of MALDI mass spectrometry for DNA analysis critically depends on mass resolution and sensitivity.

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