Sample preparation

With MALDI-TOF MS, the analysis of proteins and nucleic acids is accomplished by embedding the analyte in a crystalline structure of organic molecules, referred to as matrix. This matrix later serves as the "launching" material for mass spectro-metric analysis. The matrix-analyte co-crystal is volatilized with laser bursts. Usually, in UV MALDI, nitrogen lasers with a wavelength of 337 nm are used. The type of matrix and the laser wavelength are aligned such that during this volatilization the matrix molecules absorb the laser energy. During the last decade, for example, the matrix of choice for nucleic acid analysis by UV MALDI has been 3-hydroxy-picolinic acid. Introduction of energy into the crystal structure leads to a micro explosion, which generates a particle cloud. Analyte molecules are desorbed into the gas-phase along with the matrix molecules. Because the matrix absorbs the energy, analyte molecules remain intact and can be analyzed as intact molecules. The volatilization process is accompanied by gas-phase proton transfer reactions, which generate both analyte and matrix ions. An electric field of approximately 20 kV is used to accelerate the ions to nearly uniform kinetic energy. The ions then travel through a field-free drift region (usually 1 m length) and separate by their mass-to-charge ratio. The ions finally reach a detector, which allows the measurement of their TOF. This TOF is directly proportional to the mass-to-charge ratio. Because the MALDI process generates predominantly singly charged ions, measured signals directly represent the molecular mass of the analyte.

What sparked the interest of researchers to analyze nucleic acids by MS? The desire to establish mass spectrometric methods as a means to analyze nucleic acids was related to deficiencies in other methods commonly used: they were (and still are) based on indirect detection methods requiring a fluorescent or radioactive reporter and some of them used and still use fairly time-consuming separation methods such as gel or capillary electrophoresis (at least in DNA sequencing). In contrast, the molecular mass of the analyte is an intrinsic molecular property. It allows direct and highly accurate characterization of the underlying nucleic acid reaction product. The MALDI process is extremely fast and therefore enables high-throughput applications. Both accuracy and speed are tremendously important to set performance standards in genomic/genetic research and molecular diagnostics. Furthermore, the availability of molecular mass information also significantly improves the assay development process. Unanticipated reaction byproducts can be easily characterized on the basis of their mass, which significantly improves the ability to trouble-shoot undesired enzyme properties and enzymatic reactions, and eventually allows their optimization.

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