Quantitative matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for DNA measurements

With the tremendous worldwide efforts on cancer research, both DNA adductions and genetic mutations have been shown to be linked separately to various types of cancer diseases. In the past decade, researchers have made significant strides in the detection of various biomolecules with the use of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. In the case of measuring oligonucleotides, they are however not always easy to analyze for the following reasons. First, the strong interactions between negatively charged phosphate backbone of oligonucleotide and metal cations lead to the formation of adduct ions. Second, oligonucleotides can be easily fragmented during MALDI-TOF measurements. Third, the signal intensity of oligonucleotide from an identical sample can vary significantly. The variation in signal intensity creates a challenge for quantitative MALDI-TOF measurements. The goal of this research is to evaluate an experimental approach to circumvent the variation in signal intensity with MALDI-TOF mass spectrometry and establish the limit of quantitation for oligonucleotides. Two pure synthetic oligonucleotides were used. To ensure the MALDI-TOF measurements of both oligonucleotides have similar efficiency, the majority of their DNA sequences are identical except one of them has two extra nucleotides at the 3' end. When both oligonucleotides are mixed at the same concentration, the theoretical ratio of their corresponding signal intensities was expected to be close to one. By using this as a criterion to accept the results, together with the use of an automatic mode to measure as many as 12 replicates and the filtering of outliers, the variation in signal intensity has been significantly reduced. Various parameters of MALDI-TOF measurements were then optimized by using the above approach. Under the optimized conditions, the linear dynamic range and limit of quantitation was established to have almost two orders of magnitude that ranged from 0.20 muM to 12.5 muM. The optimized conditions did result in an improvement over the default settings for the MALDI-TOF instrument. However, the improved linear dynamic range was not equivalent to other analytical detection methods, such as fluorescence and UV absorbance. For this reason, further studies were performed and led to two new hypotheses for explaining the limitation on the linear dynamic range for MALDI-TOF MS measurements. First, the number of analyte and matrix molecules that would be desorbed by the laser pulsed is limited. Second, a limited number of ions survive in the MALDI ion source.