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

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.

Figure 1

Schematic representation of single base extension products of an A/T heterozygote.

Figure 2

Resolution of a synthetic 28mer (5′-CCA TCC ACT ACA ACT ACA TGT GTA ACA G-3′) as a function of laser energy (µJ) for selected sugar additives (4 g/l) added to the 3-HPA matrix (35 g/l). Each additive–matrix combination plus analyte was manually dispensed as five different spots and screened by manual data acquisition. From left to right are standard matrix without additive (S) and matrix with fructose, glucose, sucrose, n-acetylglucosamine, fucose, trehalose and glucosamine as additives, respectively.

Figure 3

Mass spectra of a 28mer acquired in automatic mode. (B and D) Standard 3-HPA matrix (35 g/l); (A and C) fucose-doped (1 g/l) 3-HPA matrix. (A and B) At threshold energy (3.6 µJ); (C and D) above threshold energy (7.0 µJ).

Figure 4

Average resolution as a function of laser energy (µJ) measured for a 28mer in: standard 3-HPA matrix (35 g/l) (filled circle); 3-HPA matrix (35 g/l) with fucose (1 g/l) (filled square); and 3-HPA matrix (35 g/l) with fructose (1 g/l) (filled triangle). All spectra were acquired in automatic mode. The resolution reported is an average of eight measurements from a single preparation dispensed as eight different spots.

Figure 5

Manually acquired mass spectra of two synthetic 23mer oligonucleotides (5′-GTT TCC ATT TAG TCA GTC AAC TG-3′, [M + H]⁺ 7004.6 Da; 5′-ACA TTC TTC ATA GCA TTT TAG A*A-3′, * = ribose, [M + H]⁺ 7012.6 Da) separated in mass by only 8 Da. (A) Sample prepared using 3-HPA matrix without additives (laser energy at threshold, 3.0 µJ). (B) Sample prepared by addition of an equal volume of 6 g/l fucose solution to the analyte mixture (final fucose concentration 3 g/l) (laser energy above threshold, 6.0 µJ).

Figure 6

Manually acquired mass spectra of A/T heterozygous products from a single base extension assay prepared (A) using a standard 3-HPA matrix and laser energy at the threshold level (3.0 µJ), with an expanded view of the A/T heterozygote products (B), and (C) by addition of an equal volume 6 g/l fucose solution to the analyte mixture (final fucose concentration 3 g/l) and using a laser energy above threshold (6.0 µJ).

Figure 7

Mass spectra of the A/T heterozygous products from a single base extension assay acquired in automatic mode (A) with a standard 3-HPA matrix and laser energy set at threshold level (3.0 µJ) and (B) using fucose-doped matrix (1 g/l final fucose concentration) and a laser energy above threshold (6.0 µJ).