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

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Norman H. Chiu, Assistant Professor (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/

Abstract: Sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) of DNA is critical for obtaining high quality mass spectra. Sample impurity, solvent content, substrate surface and environmental conditions (temperature and humidity) all affect the rate of matrix–analyte co-crystallization. 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 mass resolution in time-of-flight (TOF) MS as the excess energy transferred to DNA causes metastable decay. This can be overcome by either searching for ‘hot’ spots or adjusting the laser fluence. However, both solutions may require a significant amount of operator manipulation and are not ideal for automatic measurements. We have added various sugars for crystallization with the matrix to minimize the transfer of excess laser energy to DNA molecules. Fructose and fucose were found to be the most effective matrix additives. Using these additives, mass resolution for DNA molecules does not show noticeable deterioration as laser energy increases. Improved sample preparation is important for the detection of single nucleotide polymorphisms (SNPs) using primer extension with a single nucleotide. During automatic data acquisition 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. Made available courtesy of the Journal of Nucleic Acids Research and Oxford University Press. The version of record is available at: http://nar.oxfordjournals.org/content/29/19/e91.abstract

Additional Information

Publication
Language: English
Date: 2001
Keywords
chemistry, biochemistry, DNA, sugar additives, MALDI matrices, mass spectrometry