Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become an important tool in the analysis of biomolecules. The matrix-assisted laser desorption/ionization technique, or MALDI, which is based on an ultraviolet absorbing matrix, was developed in 1987.30 The method offers a rapid and accurate means for genotyping DNA samples and is a promising emerging technique.
Mass spectrometry (MS) has been widely used in forensic science in the identification of compounds, particularly illicit drugs. MS is a technique that allows the detection of compounds by separating ions by their unique mass (mass-to-charge ratios) using a mass spectrometer. The method relies on the fact that every compound has a unique fragmentation pattern (mass spectrum). The sample is ionized; the sample ions are separated based on their differing masses and relative abundance.
A typical mass spectrometer is comprised of these components:
- ion source
- mass analyzer
Biomolecules and synthetic polymers have low volatility and are thermally unstable, which has limited the use of MS as a means of characterization. These problems have been minimized through the development of MALDI-TOF MS, which allows for the mass determination of biomolecules by ionization and vaporization without degradation.
A laser beam is used to ionize the sample bound in a matrix that is used to protect the DNA from being destroyed during the process. One common matrix is 3-hydroxypicolinic acid, which when mixed with the sample and dried under vacuum conditions, leaves a recrystallized matrix.31 The DNA is homogeneously spread throughout the recrystallized matrix. The laser is pulsed onto the crystals which absorb the laser energy and are partially vaporized, carrying intact DNA into the vapor phase. The matrix crystals transfer part of their charge to the DNA, allowing for ionization, while protecting the DNA from degradation.
At the time of each laser pulse, a voltage is applied to accelerate the sample towards a time-of-flight mass analyzer. The ions enter a vacuum where they are accelerated by a strong electric field in a flight tube towards the detector. Separated in the flight tube, the smaller DNA ions arrive at the detector in a shorter amount of time than the larger ions. An analyzer measures the time-of-flight (TOF) taken for particular ions to hit the detector. Separated ion fractions arriving at the end of the flight tube are detected by a recorder that produces a signal upon the impact of each ion group. The digitized data generated from successive laser shots is summed yielding a TOF mass spectrum. The flight time of an ion is related to its mass-to-charge ratio.32,33
The use of the instrument is limited in forensic science laboratories because the method development is still underway. Despite these current limitations, MALDI-TOF MS does offer the advantages of short analysis time, automation, and accuracy.
The method has the ability to accurately determine molecular weight information without the use of allelic ladders as size standards.31 MALDI was originally designed for ionization of large polypeptides and proteins; the techniques have been modified to include the analysis of oligonucleotide single nucleotide polymorphism (SNP) DNA and other macromolecules. It has been shown to be capable of separating STR amplicons at the rate of several thousand samples per day.34 MALDI-TOF MS is amenable to automation and rapid analysis. It is not only a viable technique for STR fragment analysis, but also SNP analysis.
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