A conventional method used for DNA size measurement is gel electrophoresis. In this process, an electric field is applied to a gel medium and DNA molecules are separated by size. However, this process is slow and takes hours to separate different sizes of DNA fragments. Other physical and chemical properties of gel, such as viscosity, temperature, and temperature gradients, tend to influence the migration of DNA fragments. Alternatively, mass spectrometry has been used for several decades to measure the molecular weight of gaseous samples by mass-to-charge ratio (M/Z). In order to use a mass spectrometer for molecular weight determination, molecules of interest need to be produced in a gas phase. Owing to the practically zero vapor pressure of biomolecules at room temperature, a method to convert biomolecules from solid or liquid form into gaseous form is required. Desorption of biomolecules from solid phase using a laser beam has been pursued for decades. However, large biomolecules tend to fragment when laser desorbed, due to their strong absorption of laser photons. Results of laser desorption experiments indicated that the upper mass limit was ~5000 Daltons. Matrix-assisted laser desorption/ionization (MALDI) was developed to circumvent the problem of laser absorption-induced dissociation (1). With MALDI, laser energy is absorbed by matrix molecules instead of the biomolecule so that no fragmentation of the biomolecule occurs. Because mass spectrometry is based on the detection of gas phase ions, a means to produce ions is also needed. In the MALDI process, positive or negative biomolecular ions are produced by attaching or detaching a proton to/from a biomolecule. Once the ions have been produced, various sizes of ions may be separated by electric or magnetic fields. The mass resolved ions are then accelerated to impinge on a charged particle detector such as an electron multiplier or a microchannel plate (MCP). When one ion impacts an electron multiplier, as many as 106 electrons can be produced after several stages of multiplication, thus making it possible to detect even a single charged particle.
Owing to its relatively simple principle of operation, which is somewhat analogous to a gel electrophoresis device, a time-of-flight mass spectrometer (TOF-MS) is most
From: Methods in Molecular Biology, vol. 217: Neurogenetics: Methods and Protocols Edited by: N. T. Potter © Humana Press Inc., Totowa, NJ
often used for biomolecule detection. A TOF-MS is a device in which ions of different masses are given the same energy and are allowed to traverse a field-free distance. Because of their different velocities, the ions of differing masses arrive at the end of the field-free distance at different times. In order to achieve high resolution in mass spectra, the ions need to be produced in a very short time interval and in a very small volume. Thus, ionization by a short-duration pulsed laser is an ideal choice for a TOF-MS. One major advantage of a TOF-MS over other types of mass spectrometers is its ability to measure all of the masses in a sample in a very short period of time. Another is its capability of measuring ions with very high molecular weights. In principle, there are no limitations on the size of molecules that can be measured by a TOF-MS.
In 1987, Hillenkamp and his coworkers (1) discovered that large protein molecular ions can be produced without much fragmentation by laser desorption, if these biomolecules are mixed with smaller organic compounds, which serve as a matrix for strong absorption of laser photons. This process is now called matrix-assisted laser desorption (MALD). The typical preparation technique for MALD is to dissolve biomolecular samples in solution, then prepare another solution of small organic molecules, such as 3-hydroxypicolinic acid. These two solutions are subsequently mixed and a small amount is placed on a metal plate to dry. After the crystallization of the sample, the sample plate is placed in the mass spectrometer for analysis. The molar ratio of matrix to analyte is typically more than 1000 to 1. During the MALD process, matrix molecules strongly absorb the laser energy and become vaporized, carrying the large interspersed biomolecules along with them during the fast vaporization process. Large gas-phase biomolecules can be produced without fragmentation, which is probably due to minimal direct absorption of laser energy; thus, "soft" desorption can be achieved. The meaning of "soft" here indicates no breaking apart of molecules. However, it was found that protein parent ions are also produced during the MALD process in addition to the expected neutral molecules. Thus, these desorbed ions can be directly detected by a mass spectrometer. The process involving ionization and matrix-assisted laser desorption at the same time is abbreviated MALDI (namely, matrix-assisted laser desorption and ionization). The mechanism of ion production has been speculated by many researchers to involve proton transfer. The proton attachment to a biomolecule leads to the production of a positive ion, namely (M + H)+. The proton transfer from biomolecule to matrix molecule produces negative ions, namely (M - H)-.
Since the discovery of MALDI, many research groups have succeeded in using it to measure various proteins and large organic compounds. MALDI has also been applied to DNA segments. Initially, success was limited to small DNA detection and gaining some understanding of the mechanism of the MALDI process. Then, Wu et al. (2) discovered 3-hydroxypicolinic acid is a good matrix for mixed-base oligonucleotide and succeeded in detecting oligonucleotides of 67 bases. With the development of an instrument to give high ion energy and the use of new matrices, we reported the first measurement of detecting longer oligonucleotides (500 bp) with MALDI (3). That confirmed that MALDI can be used to measure long DNA for various applications.
MALDI is emerging as a new technology for the rapid, reliable, and inexpensive detection of genetic polymorphisms and mutations (4). The separation time needed for MALDI can be shorter than a few hundred microseconds, compared to hours for conventional gel analysis and minutes for capillary gel electrophoresis. Since MALDI
detection of biomolecules is based on molecular mass, there is no need to exogenously label biomolecules, which can significantly reduce both time and cost. In addition, there is no concern for identification ambiguity resulting from band compression, reiterated nucleotide sequences or high GC content. Collectively, these advantages make MALDI mass spectrometry an attractive methodology for automated, high-throughput DNA genotyping.
We have utilized MALDI for DNA sequencing (5-7), genotyping for cystic fibrosis CFTR mutations (8,9), single nucleotide polymorphism (SNP) detection (9), short tandem repeat (STR) genotyping for forensic applications (10), gender determination (11), and multiplex hybridization detection (12). Recently, we have utilized MALDI for the analysis of trinucleotide repeat (TNR) containing genes and demonstrated its validity for the accurate genotyping of expanded CAG alleles associated with both Huntington's disease (HD) and Dentatorubral pallidoluysian atrophy (DRPLA), two autosomal dominant neurodegenerative disorders (13). In this chapter, we outline the methodology for the application of MALDI for the detection and quantitation of TNR containing genes and illustrate its utility for the accurate genotyping of this type of DNA mutation.
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