Sugar additives for MALDI matrices improve signal allowing the smallest nucleotide change (A : T) in a DNA sequence to be resolved

Sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of DNA Is critical for obtaining high quality mass s pectra. Sample Impurity, solvent content, substrate surface and environment al conditions (temperature and humidity) all affect the rate of matrix-anal yte cocrystallization. As a result, laser fluence threshold for desorption/ ionization varies from spot to spot. When using 3-hydroxypicolinic acid (3- HPA) as the matrix, laser fluence higher than the threshold value reduces m ass resolution In time-of-flight (TOF) MS as the excess energy transferred to DNA causes metastable decay. This can be overcome by either searching fo r 'hot' spots or adjusting the laser fluence. However, both solutions may r equire a significant amount of operator manipulation and are not ideal for automatic measurements. We have added various sugars for crystallization wi th the matrix to minimize the transfer of excess laser energy to DNA molecu les. Fructose and fucose were found to be the most effective matrix additiv es. Using these additives, mass resolution for DNA molecules does not show noticeable deterioration as laser energy Increases. Improved sample prepara tion is Important for the detection of single nucleotide polymorphisms (SNP s) using primer extension with a single nucleotide. During automatic data a cquisition It Is difficult to routinely detect heterozygous A/T mutations, which requires resolving a mass difference of 9 Da, unless a sugar Is added during crystallization.