Plant Genome III Conference Plant Genome III Conference
Mass spectrometers have only recently been able to access mass ranges that might be suitable for oligonucleotide sequencing. The combination of matrix-assisted lmr desorption/ionization (MALDI) and time-of-flight (TOF) mass spectrometry is perhaps the most promising approach. Using this combination, proteins molecular weights of up to 500 kDa have been measured; however, oligonucleotides in the same mass range have proven to be more intractable since the presence of multiple charge centers on phosphate groups results in reduced desorption efficiency, while decomposition during the mass measurement results in loss of mass resolution.
At the same time, current technology would appear to enable detection of oligomers up to the 100mer with better dm unit base resolution. While this involves oligonucleotide segments that arc shorter than those analyzed by gel electrophoresis, the decrease in analysis time resulting from direct mass analysis of oligonucteotide ladders (without electrophoretic separation) should increase the overall sequencing speed. In addition, mass resolution is currently being improved using an instrument equipped with a curved-field reflection that has been developed in our laboratory, suggesting that large DNA segments will be accessible.
This paper reviews the principles of time-of-flight measurements, the mechanisms by which MALDI desorbs and ionizes macromolecules, and a variety of approaches (UV laser desorption; IR desorption from frozen matrices, etc.) that have been utilized to date.